transfection of nucleic acids

Table of contents :

• transfected gene expression to protein
• classical gene therapy
• a few strategies of non conventional gene therapy
• DNA vaccines
• biomolecular computer
• early  research focused on laboratory-scale, human-operated computers for complex computational problems
• recently, simple molecular-scale autonomous programmable computers were demonstrated allowing both input and output information to be in molecular form. Such computers, using biological molecules as input data and biologically active molecules as outputs, could produce a system for 'logical' control of biological processes. An autonomous biomolecular computer that, at least in vitro, logically analyses the levels of mRNA species, and in response produces a molecule capable of affecting levels of gene expression has been described. The computer operates at a concentration of close to a trillion computers per microlitre and consists of 3 programmable modules:
• a computation module, that is, a stochastic molecular automaton
• an input module, by which specific mRNA levels or point mutations regulate software molecule concentrations, and hence automaton transition probabilities
• an output module, capable of controlled release of a short single-stranded DNA molecule.
This approach might be applied in vivo to biochemical sensing, genetic engineering and even medical diagnosis and treatment. As a proof of principle the computer was programmed to identify and analyse mRNA of disease-related genes associated with models of SCLC and prostate cancer, and to produce a single-stranded DNA molecule modelled after an anticancer drug^ref. So far, the computer only works in the confines of a finely balanced salt solution, and there are many hurdles to overcome before it can be applied to real disease. It is necessary to ensure that the computers will survive inside a biological setting, will not provoke an immune response and will be safe to use. They would also need to be far more complex than the prototype, which recognizes only messenger RNAs related to cancer. And they would need to deliver a wide variety of drugs, not just DNA therapies. They would need to be tested in cell suspensions, tissue cultures, simple organisms, mammals and finally humans.
• genetic enhancement : enhance traits and capacities of ...
• humans : genetic doping : muscles transfected with IGF-1 can be unmasked with gene markers, MRI or muscle biopsy
• cattle : gene markers for marbling (the fat within the muscle in a cut of meat – a valued trait which improves the eating quality of beef and is highly sought after by many of Australia’s high value export markets) and tenderness : 5 genes, including receptor for vitamin A, assayed with GeneSTAR^® Feedlot
• crops :
• creation of transgenic cells, tissues or organisms. In 2003, the EU lifted a 1998 ban on genetically modified crops but enacted strict labelling and traceability rules for products with genetically modified ingredients. The EU has approved about 12 genetically engineered crops, including certain types of corn, canola and soy beans. GM biologicals could be useful for ...
• engineering
• Gene oscillators : synthetic gene network for entraining and amplifying cellular oscillations^ref. The synchronization properties of a coupled system (oscillator to a periodic process that is intrinsic to the cell) and how the oscillator can be constructed to yield a significant amplification of cellular oscillations. The ability to couple naturally occurring genetic oscillations to a synthetically designed network could lead to possible strategies for entraining and/or amplifying oscillations in cellular protein levels
• a synthetic oscillatory network of transcriptional regulators^ref : networks of interacting biomolecules carry out many essential functions in living cells, but the 'design principles' underlying the functioning of such intracellular networks remain poorly understood, despite intensive efforts including quantitative analysis of relatively simple systems. A synthetic network to implement a particular function was designed using 3 transcriptional repressor systems that are not part of any natural biological clock to build an oscillating network, termed the repressilator, in Escherichia coli. The network periodically induces the synthesis of green fluorescent protein as a readout of its state in individual cells. The resulting oscillations, with typical periods of hours, are slower than the cell-division cycle, so the state of the oscillator has to be transmitted from generation to generation. This artificial clock displays noisy behaviour, possibly because of stochastic fluctuations of its components. Such 'rational network design may lead both to the engineering of new cellular behaviours and to an improved understanding of naturally occurring networks
• design of artificial cell-cell communication using gene and metabolic networks^{ref1, ref2} : artificial transcriptional networks have been used to achieve novel, nonnative behavior in bacteria. Typically, these artificial circuits are isolated from cellular metabolism and are designed to function without intercellular communication. To attain concerted biological behavior in a population, synchronization through intercellular communication is highly desirable. A gene-metabolic circuit that uses a threshold concentration of a common metabolite (acetate) to achieve tunable artificial cell-cell communication (induce gene expression by acetate kinase and part of the nitrogen-regulation 2-component system) has been constructed. As one application of the cell-cell communication circuit an artificial quorum sensor was created. Engineering of carbon metabolism in Escherichia coli made acetate secretion proportional to cell density and independent of oxygen availability. In these cells the circuit induced gene expression in response to a threshold cell density. This threshold can be tuned effectively by controlling DpH over the cell membrane, which determines the partition of acetate between medium and cells. Mutagenesis of the enhancer sequence of the glnAp2 promoter produced variants of the circuit with changed sensitivity demonstrating tunability of the circuit by engineering of its components. The behavior of the circuit shows remarkable predictability based on a mathematical design model
• bio-engineered household pets
• the normally black-and-silver zebra fish (Danio rerio) inserted with genes from sea anemones or jellyfish to turn them red or green, and glow under black or UV lights (GloFish^®). In USA federal agencies have decided they have no jurisdiction over a bio-engineered household pet that is not intended for consumption, but California has blocked sales : the altered fish tolerate cold less than natural zebra fish, and they could not survive in California waters.
• cats genetically engineered to be hypoallergenic will be produced by Allerca Inc. by 2007 using RNA interference to "silence" a gene in cats that produces the irritant, which is excreted through saliva and the skin
• gene targeting is accomplished using embryonic stem cells (ESCs) in the mouse but has been successful, only using primary somatic cells followed by embryonic cloning, in other species. Gene targeting in somatic cells versus ESCs is a challenge; consequently, there are few reported successes
• conditional knockout technology
• double targeting to produce homozygotes. The sequential gene targeting system alleviates the need for germline transmission for complex genetic modifications and should be broadly applicable to gene functional analysis and to biomedical and agricultural applications^ref
• assessing the role of proteins in biological phenomena
• different levels of transgene expression
• ATP analogue-sensitive kinase alleles (ASKA) technology

phage display : the protein coding sequence (cds) is inserted between the leader peptide cds and the protein III (pIII) NTD cds of Enterobacteria phage M13, subspecies fd (fd-Tet^R-DOG1 phage vectors and amp^R pHEN1 phagemid vectors) : 3-5 molecules of pIII are expressed per virion surface. The recombinant phage that are produced with the aid of M13KO7 helper phage contain a single-strand copy of the phagemic DNA encoding a specific gene (e.g. scFv), displayed on the phage tips as fully functional fusion proteins. Selection and enrichement of the desired phage is accomplished by panning against the target ligand either on a solid phase (=> ELISA with HRP/antiM13 conjugate) or in solution using NHS-LC-biotin-labeled ligand (=> ligand/protein complexes are then captured using streptavidin coupled to a crosslinked beaded agarose : this method allows for not only easy manipulation of ligand concentration but, based on the quantity of ligand used, selection of protein that exhibit high affinities or kinetics of dissociation). After washing, only the virions infected with the phage carrying the specific protein remain attached to the plate and can be recovered through eluition at pH 2 (this phage resists such pH !). The resultant ligand-positive clone can be amplified by PCR and used to infect Escherichia coli HB2151 to produce soluble protein (expressed in Escherichia coli periplasm or secreted into the culture medium), and even further purified from other E.coli proteins by affinity chromatography and used to infect carrier Escherichia coli. New methodologies allow the screens to be done directly against cell surface proteins, first in tissue culture^ref and later in vivo^ref.A phage chimera with 2 display sites that retain their functionality in vivo simultaneously presents a peptide of interest on the conventional site, as a fusion with the minor coat protein pIII at the tip of the filamentous body, and a streptavidin-binding peptide on the major coat protein pVIII that is distributed throughout the outer surface of the phage particle. This bifunctional phage can thus link a peptide to any moiety that can be bound via streptavidin, eliminating the need to chemically conjugate the selected peptide to the functional end. Using the chimera to couple streptavidin-coated quantum dots to a peptide that targets cancer cells (through its binding to a_v-integrins), the group demonstrated the feasibility of imaging cancer cells in mice. The problem with quantum dots is that in vivo they quench very rapidly : by using pVIII, one could express the streptavidin-binding peptide throughout the stringy body of the phage, have many more copies, and thus have a phage particle that is very bright. The approach sounds deceptively simple but was not guaranteed success, as the combination of 2 display sites could also multiply each of their limitations, mainly the low density of the peptides of interest compared to the total phage surface and the low affinity of the phage-target complexes. However, upon extensive testing, the 2 binding moieties appeared functional and the chimera has lived up to expectations. The in vivo imaging application is certainly exciting, but the chimera also opens up the door to a tantalizing variety of in vitro applications. As proof of principle, the authors were able to harness the cell-targeting capacity of a peptide by association to an array of streptavidin-coated functional supports: tissue culture plastic, magnetic beads, biosensor chips and fluorescent beads. Among the exciting possibilities explored by the authors is the combination of bifunctional display with real-time binding measurements obtained using surface plasmon resonance. The bifunctional phage can be immobilized on a streptavidin-coated sensor chip, exposing the peptide of interest as cells are passed over the sensor surface and their binding is measured in real time. The technology still has limitations and the signal is quite variable. However, experimenting with the new BIAcore machines, Arap and Pasqualini are confident of success and envision that the ability to translate phage display into protein-protein interactions that can be reliably quantified will allow important applications^ref. Phage technology was first optimized for display of scFv^ref and Fab^ref fragments derived from either immunized donors^ref or synthetic (naïve) repertoires^{ref1, ref2, ref3}. Phage library construction has been the subject of several excellent reviews^{ref1, ref2,ref3} and the largest commercial phage libraries and their methods of construction are available on the following websites:
• CAT
• Morphosys
• BioInvent
• Dyax
Recombination technologies can avoid some of the limitations evoked by bacterial transformation^ref (Zyomyx). Efficient affinity improvements have been
obtained through the development of innovative affinity maturation methods^{ref1, ref2, ref3, ref4} that have evolved from the original methods^ref such as chain shuffling^ref or use of mutator cells^{ref1, ref2}.
• imaging of biological phenomena
• gene marking (e.g. to define the origin of tumor relapse in autologous HSC transplantation (graft or recipient) and the efficiency of stem cells gene transfer)
• Yoda, a mouse genetically modified in his pituitary and thyroid glands and with reduced insulin production (hence a third smaller in body size than an average mouse and very sensitive to cold) has celebrated his fourth birthday (the human equivalent of about 136 years), making him the oldest of his kind (an average lab mouse lives slightly > 2 years) : he is still mobile, sexually active and looking good. Yoda lives in a carefully maintained lab with roughly 100 other male geriatric mice being used for a lifespan study. Yoda's cage mate, Princess Leia, is a much larger female who uses her body warmth to keep the dwarf mouse from freezing to death.
• resistance to transmissible diseases
• PrP^Sc-/- cows created by researchers in the USA (Hematech) and Japan  will be used for research into the possibility of engineering cattle to produce human antibodies for medical applications. Their BSE-free status will alleviate health fears over the human consumption of cow products. But the technology seems unlikely to end up on our dinner plates, as many consumers refuse to eat genetically modified food and are opposed to animal cloning^ref
• production of proteins for human use :

Expression systems :
• prokaryotic expression systems : produced proteins often differ in structure (e.g. in glycosylation patterns of glycoproteins (some hormones, immunoglobulins, ...)) => they may induce immune responses or be less stable when injected into the recipient individual. Posttranslation modification may be achieved only by co-expression of processing enzymes. Some proteins expressed in large amounts precipitate inside the cell and form inclusion bodies : for extraction, it is unavoidable firstly to denature the proteins using agents like urea or guanidinium hydrochloride and to renature them carefully. Alternatively, a reduction of the expression level can be envisaged by using a weaker or an inducible promoter.
• Escherichia coli expression systems : proteins larger than 300 amino acids are very poorly expressed, while for proteins smaller than 100 amino acids the addition of a fusion partner (which in most cases appears to stabilize the protein of interest) is recommended (anyway the fusion partner may by itself be antigenic and give rise to false-positive results). This system requires high purity level because antibodies against Escherichia coli proteins, which would give false positive results in antibodies testing, are frequent in humans.
• overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. The unique features of the cspA gene have been applied to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock^ref
• eukaryotic expression system : use or viral vectors with a lytic infection cycle obviate the precipitation of the overexpressed protein and formation of inclusion bodies.
• yeast cell systems : yeast glycosylation is of the high-mannose type, which confers a short in vivo half-life to the protein and may render it less efficacious or even immunogenic. Several ways of humanizing yeast-derived glycoproteins have been tried, including enzymatically modifying proteins in vitro and modulating host glycosylation pathways in vivo.
• plant cell systems : the use of whole plants for the synthesis of recombinant proteins has received a great deal of attention recently because of advantages in economy, scalability and safety compared with traditional microbial and mammalian production systems. However, production systems that use whole plants lack several of the intrinsic benefits of cultured cells, including the precise control over growth conditions, batch-to-batch product consistency, a high level of containment and the ability to produce recombinant proteins in compliance with good manufacturing practice. Plant cell cultures combine the merits of whole-plant systems with those of microbial and animal cell cultures, and already have an established track record for the production of valuable therapeutic secondary metabolites. Although no recombinant proteins have yet been produced commercially using plant cell cultures, there have been many proof-of-principle studies and several companies are investigating the commercial feasibility of such production systems.


• insect expression systems : insect cells present several comparative advantages to mammalian cells, such as ease of culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus. The culture of insect cells in the absence of serum is reaching maturity, and promising serum substitutes (hydrolysates, new growth and production-enhancing factors) are being evaluated. Proteolysis is a problem of the BEVS system due to its lytic nature, and can, therefore, be a critical issue in insect cell bioprocessing. Several cell- or baculovirus proteases are involved in degradation events during protein production by insect cells. In the baculovirus expression vector system (BEVS) Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), which infects lepidopteran Spodoptera frugiperda (fall armyworm) Sf9 or Estigmene acrea Ea4 cells. Due to the fundamental nature of insect glycoprotein processing pathways, this system is typically unable to produce recombinant mammalian glycoproteins with full extent oligosaccharide side chains, a phenomenon believed to be due to saturation of the processing pathway during overexpression : the major processed O- and N-glycan species found on these glycoproteins are (Galb1,3)GalNAc-O-Ser/Thr and Man₃(Fuc)GlcNAc₂-N-Asn, respectively. Anyway this hurdle can be bypassed by co-expression of human glycosyl-transferases (e.g. GlcNAc transferase II (GnT2), b1,4-galactosyltransferase (b14GT), and a2,6-sialyltransferase (a26ST) by a single recombinant baculovirus. However, the ability or inability of insect cells to synthesize and compartmentalize sialic acids and to produce sialylated glycans remains controversial. This is an important issue because terminal sialic acid residues play diverse biological roles in many glycoconjugates. While most work indicates that insect cell-derived glycoproteins are not sialylated, some well-controlled studies suggest that sialylation can occur. Cultivation in large 40-1 bioreactor allows sufficient batch sizes : a recombinant His₆ affinity tag facilitates its purification, using the immobilized metal affinity chromatography (IMAC) technique. This affinity purification method allows mild elution conditions that preserve the structure of the protein. Automation of both fermentation processing times and purification processes shortens processing times considerably and therefore, helps to protect the structure and function of the recombinant human proteins. MultiBac is a simple and versatile system for generating recombinant baculovirus DNA to express protein complexes comprising many subunits by using transfer vectors containing a multiplication module that can be nested to facilitate assembly of polycistronic expression cassettes, thereby minimizing requirements for unique restriction sites. The transfer vectors access a modified baculovirus DNA through Cre-loxP site-specific recombination or Tn7 transposition. This baculovirus has improved protein expression characteristics because specific viral genes have been eliminated. Gene insertion reactions are carried out in Escherichia coli either sequentially or concurrently in a rapid, one-step procedure. This system is useful for both recombinant multiprotein production and multigene transfer applications^ref.

Web resources : Protein Sciences Corp.
• mammalian cell systems : recently, the productivity of mammalian cells cultivated in bioreactors has reached the gram per liter range in a number of cases, a more than 100-fold yield improvement over titers seen for similar processes in the mid-1980s.
• Vaccinia virus in Chinese hamster ovary (CHO) cells. It is easy to grow, the introduction of foreign genes is relatively simple and, due to the size of the vaccinia genome, it can accept large pieces of foreign DNA
To create human-style glycoproteins, some researchers are breeding genetically engineered plants or cattle that might secrete the molecules in their leaves or milk
Kinds of protein :
• enzymes to increase, decrease, or add specific proteins ... :
• .... that are used industrially :
• GM plants for industrial uses
• ... in the edible parts (GM food)
• tailoring foods to better meet our needs
• amino acid composition of proteins in basic grains
• specific vitamin contents such as vitamin A or vitamin E
• specific components such as caffeine or phytic acid conceivably can be eliminated in the source plant, negating the need for processing steps that add cost and that lessen flavor and nutrition
• resistance to ...
• pests, diseases
• environmentally benign pesticides
... of certain ...
• crops
• animals
• GM mice strain transgenic for fatty acid desaturase (fat-1), from the roundworm Caenorhabditis elegans, converts w-6 fatty acids into the healthier w-3 version (mammals can't ordinarily do this), resulting in an abundance of n-3 and a reduction in n-6 fatty acids in the organs and tissues of these mice, in the absence of dietary n-3^ref. This technology might be adapted to enrich n-3 fatty acids in animal products such as meat, milk and eggs
• Herman the Bull, the world's first farm animal carrying a human gene. A human gene was spliced into Herman's genetic code while in an early embryonic stage in 1990 in the Netherlands, in the hope that milk produced by his female offspring would bear a human milk protein. The process was cutting edge at the time, but has since been refined and is now commonly used. The experiment was only a partial success. Milk from Herman's descendants contained the proteins, but at such low levels that it was not commercially worthwhile to extract them. He was put down on April 2, 2004 because he was suffering from a form of arthritis, unrelated to his genetic manipulation. He was 13, not very old for a bull. 2 cloned cows, Holly and Belle, kept him company in his final years.
See also risks for health.
• antigens (Ags) for
• autoantibody testing
• subunit vaccines
• purified molecules
• edible (oral) vaccines
• rMHC class I molecules for DNA vaccines against virus infection or tumors
• single chain trimers of MHC class I molecules consisting of an antigenic peptide-spacer-b₂- microglobulin-spacer H chain assemble efficiently, mantain their covalent structure and are unusually stable at the cell surface. Consequently, these constructs are at least 1000-fold less accessible to exogenous peptide than class I molecules loaded with endogenous peptide, and they are potent stimulators of peptide-specific CTL and Abs.
• recombinant monoclonal antibodies (r-mAbs) : normal plasma cells live only for 1-2 weeks in culture^{ref1, ref2, ref3, ref4}. Currently, > 100 mAbs are in clinical trials, and 18 MAbs are approved for therapeutic use in the United States, and the global therapeutic MAb market is worth $5.4 billion in 2003, a number that is predicted to triple in the next five years. By one estimate, MAbs will account for 32% of all revenues in the biotech market by 2008 (Monoclonal Antibody Therapies 2004: Entering a New Competitive Era Minneapolis: Arrowhead Publishers 2004). MAbs also contribute to the in vitro diagnostics market expected to be worth some $34 billion in 2005, according to a market analysis by Theta Reports^ref. < 1% of mAbs are function blocking
• 1895 2 French physicians attempted a radical departure from the standard cancer treatment regimen. Instead of surgery, Charles Richet and Jules Hericourt administered an antiserum derived from dogs to patients with advanced cancer.1 Some patients improved, although significant immunogenicity problems occurred. Lacking specificity and purity, the formulation cured no one (J Hericourt, C Richet  "'Physiologie Pathologique' – de la serotherapie dans la traitement du cancer," Comptes Rendus Hebd Seanc Acad Sci 1895, 121: 567)
• 1975 : César Milstein and Georges Kohler at the Medical Research Council's (MRC) Laboratory of Molecular Biology (LMB) discover how to isolate mAbs using hybridoma cells
• 1980 : MRC uses expertise to help establish Celltech, marking the start of the UK biotech sector
• 1982 : first mAb specific for human keratin produced
• 1983 : first mAb diagnostic introduced, for Chlamydia
• 1984 : a sensitive mAb-based diagnostic assay for TSH introduced into routine clinical biochemistry. Milstein and Kohler awarded Nobel Prize
• 1986 : Greg Winter and Michael Neuberger at LMB pioneer techniques to humanize mouse antibodies
• 1987 : first chimeric and humanized antibodies enter clinical trials
• 1986 : FDA approves the first therapeutic mAb, murine-developed muronomab, for acute organ rejection
• 1988 : Daniel Jay at Tufts University develops CALI
• 1989 : Neuberger and Marianne Bruggermann at the Brabaham Institute, Cambridge, genetically engineer mice to produce human antibodies
• 1990 : Winter develops phage production of human antibodies
• 1994 : FDA approves abciximab for hemostasis, the first approved therapeutic chimeric mAb
• 1997 : FDA approves rituximab, the first mAb for cancer (non-Hodgkin lymphoma) and daclizumab for immune diseases, the first approved therapeutic humanized mAb
• 1998 : FDA approves infliximab for rheumatoid arthritis and other autoimmune diseases. Global sales of US$1.6 million in 2002 make infliximab the most successful mAb therapeutic mAb to date
• 2002 : FDA approves adalimumab, the first fully human therapeutic mAb, for rheumatoid arthritis. Global therapeutic mAb market worth US$ 5.4 billion
• 2004 : currently 18 mAbs approved for therapeutic use in the USA : 3 murine, 5 chimeric, 9 humanized, 1 human. 9 of these approved in EU
Mechanisms of production :
• autonomously diversifying library (ADLib) using the chicken DT40 B-cell line undergoing gene conversion, a type of homologous recombination that primarily contributes to diversification of the immunoglobulin gene. The gene conversion frequency at the immunoglobulin locus is increased by treating DT40 cells with a histone deacetylase inhibitor, trichostatin A (TSA), thereby generating diversity at the immunoglobulin locus in the majority of treated cells. This indicates that TSA treatment accelerates the autonomous diversification of surface IgMs on DT40 cells. This effect was used to select DT40 cells producing specific antibodies with antigen-conjugated magnetic beads. This selection system enables the quick establishment (1 week from a diversifying library) of various clones producing monoclonal IgMs with enough specificity and affinity for immunological assays, and is applicable to various biotechnologies including rational protein design^ref.
• iterative antigen-mediated affinity maturation of B-cell lines that constitutively hypermutate their immunoglobulin V genes during culture can be exploited to generate antibodies in vitro.
• hypermutating human B-cell line Ramos expressing IgM of unknown specificity, descendants that exhibit stepwise improved binding to antigen can be derived. Binding is initially conferred by mutations in CDRs, but maturation is due to strategic FRs mutations.
• hypermutating chicken B-lymphoma line : a more powerful system owing to its rapid proliferation, high rate of mutation accumulation, and genetic tractability. Selection is initiated at an exceedingly low affinity threshold, but antibodies can be delivered with nanomolar affinities.
The strategy could prove useful for in vitro generation of antigen-specific monoclonal antibodies and may be extendable to the maturation of other protein-ligand interactions^ref.
• murine mAb from hybridomas (Georges Kohler and Cesar Milstein, 1975^ref) (=> when administered in humans elicit human anti-mouse antibodies (HAMA)) : rats (Rattus norvegicus), mice (Mus musculus) or guinea pigs (Cavia porcellus) are immunized with the selected antigen
• recommended mouse inoculation routes and maximum injection volumes
• subcutaneous (s.c.) : 0.2 mL
• intraperitoneal (i.p.) : 0.5 mL
• intravenous (i.v., tail) : 0.2 mL
• recommended doses of immunogen
• soluble or membrane proteins : 10–100 mg
• nucleic acids : 200 mg
• eukaryotic cells : 2–20 × 10⁶
• bacterial cells : 50 mg of protein
• viruses : 10⁷ particles × 3 weekly
• fungal antigens : 20–100 mg
Spleen or lymph node is harvested and B cells separated. By using ...
• polyethylen glycol (PEG)
• inactivated Parainfluenzavirus 1 (Sendai virus))
... these normal HGPRT(rat, mouse, or guinea pig)⁺ Ig⁺ rat, mouse or guinea pig plasma cells are fused using PEG of mol.wt. 4000 at 20°C to a suitable rat, mouse or guinea pig transformed Ig^-  myeloma B-cells with one or both of the following mutations :
• HGPRT^- (they can be produced by mutagenesis and selection in thioguanine or azaguanine, which is metabolyzed by HGPRT to nonfunctional purines)
• TK^- (they can be produced by mutagenesis and selection in 5-bromodeoxyuridine, which is metabolized by TK to a light-sensitive form, causing cell death)
Most commonly used murine myeloma cell lines are :
• X63.Ag8.653
• SP2/O
• NS1
• P3U1
Myeloma cells (instead of any other tumor cell) are used as fusion is easier and hybrid is more stable than when different cytotypes are used.
The hybridoma cells are positively selected by growth in HAT medium and expand in very-low density cultures (1 clone per well) : hybridoma clones with the desired antigenic specificity are selected by RIA or  ELISA, subcloned in semi-solid agar or by limiting diluition assay (LDA), freezed and thawned at need, producing mAbs in a mass growth culture or ex vivo. mAbs can be used directly as supernatants or ascites fluid experimetally but are purified for clinical use.
Web resources :
• Hybridoma Data Bank (HDB)
• Developmental Studies Hybridoma Bank (DVHB) at University of Iowa
• Journals :
• Hybridoma (impact factor 2001 : 0.698)
• chimaeric or partially humanized mAbs (=> when administered in humans they can still elicit human anti-chimeric antibodies (HACA)). Humanization can occur as :
• V / C mAbs : V regions from rodent and C regions from Homo
• CDR-associated mAbs : CDRs from rodent and the rest (90-95%) from Homo. Murine cells are transfected with a complete haplotype for V regions or an already rearranged human VDJ region (PDL and Aeres Biomedical)
• SDR-associated mAbs : SDRs from rodent and the rest from Homo. Even then, amino acid changes that would be predicted to have little effect on the antibody could unexpectedly abrogate antibody function.
• deimmunization by modification of the polypeptide sequence to remove T-cell-stimulating epitopes (Biovation).
Anyway ...
• ... mAb glycosylations are not the same than in the final recipient species, because of species-specific glycosyl transferases.
• ... in vivo specificity is not absolute due to affinity maturation, that allows recognition of Ags even different from the initial intended one.
• completely humanized mAbs (=> when administered in humans they can still elicit human anti-humanized antibodies (HAHA))
• human B lymphocytes from lymph node are immortalized by HHV-4 / EBV transformation.
• mouse-human heterohybridomas : mitogen-stimulated B lymphocytes from lymph node of mice immunized against the target antigen are fused to a human myeloma cell line. Lower genetic stability due to great phylogenetic distance
• SPAZ4
• SA2
• SA3
• human-human hybridomas : mitogen-stimulated B lymphocytes from lymph node of human immunized against the target antigen are fused to a human myeloma cell line :
• LICR-LON-HMy2 / LICR-2
• 5KO-007
• ARH77
• FU-266
• GM1500
• GM4672 is an IgG₂ k-producing lymphoblastoid cell line derived from a patient with multiple myeloma. The V_H region belongs to the V_H4 family and is most homologous with the V_H71-2 (87.9%), DK1, and J_H4 germline genes. The entire heavy chain V region contained 41 mutations in 36 codons and included 11 N nucleotide additions flanking the D region. GM 4672 V_k region contained a V_k1 gene rearranged with a JK4 gene. The V_k germline gene used by GM 4672 light chain was not identified but showed the most homology with Vb' germline gene (87.7%). When compared to Vb' and J_k4 genes, there were 37 mutations in 30 codons with evidence of antigen selection as determined by the replacement to silent mutation ratio in the complementarity-determining regions. The high frequency of mutations in the V region genes of GM 4672 is comparable to the sequences of other myeloma proteins^ref.
• 6TG-A1 2
Human-human hybridomas have the following difficulties :
• high tendency of multikaryon formation
• preponderance of low affinity IgM mAbs
• difficulty or ethical problems of finding humans immunized against the target antigen
• human antibody-display libraries were used to transform a mouse antibody in vitro into a fully human derivative (D2E7), which is likely to be the first FDA-approved fully human anti-inflammatory antibody.
• XenoMouse™ technology : XenoMouse strains are genetically engineered mice resulting from successive breedings in which the murine IgH and Igk loci have been first inactivated (by HR-mediated deletion of J_H and C_k regions, respectively) and then functionally replaced by their megabase-sized, germline-configured, human Ig counterparts on yeast artificial chromosome (YAC) transgenes through yeast spheroplast-ES cell fusion technology. These human Ig transgenes carry the majority of the human variable repertoire and can undergo class switching from IgM to IgG isotypes. The large and complex human V repertoires on the YAC transgenes support development of a large B cell population and the formation of a broad and diverse primary immune repertoire. The human genes are compatible with mouse enzymes mediating class switching from IgM to IgG as well as somatic hypermutation. The immune system of the XenoMouse strains recognizes administered human antigens as foreign, with a concomitant strong human humoral immune response consisting of the development of human antibodies that have undergone mouse somatic hypermutation and selection to relatively high affinity^ref. Antibodies can be recovered by classic hybridoma technology or, for more efficient affinity enhancement, by in vitro display and selection technologies, to produce IgG mAbs with sub-nanomolar affinities for human antigens^ref. Abgenix of Fremont, Calif., was the first company to turn an ordinary mouse into a human antibody factory. Called XenoMouse, it is a transgenic animal in which native antibody genes have been replaced with their human counterparts. Abgenix has used the platform both for internal drug development and in partnership with other drug developers, including Amgen in Thousand Oaks, Calif., Human Genome Sciences in Rockville, Md., and Chiron in Emeryville, Calif. According to Abgenix's Web site, 11 XenoMouse-generated antibodies have moved into clinical trials, including ABX-EGF (panitumumab), an anticancer drug targeting the epidermal growth-factor receptor. Developed in partnership with Amgen, panitumumab is currently in Phase II trials for metastatic colorectal cancer. Medarex, based in Princeton, NJ, has also developed a fully human transgenic mouse platform. Medarex's HuMab-Mouse technology allows for faster production of fully human MAbs : the company and its partners have > 150 fully human MAbs in development, including MDX-010. E.g. anti-MK-1 (Ep-CAM)^ref
• antibody fragments : for cytokine inactivation, receptor blockade or viral neutralization, the Fc-induced effector functions are often unwanted. In comparison with whole antibodies, small antibody fragments exhibit better pharmacokinetics for tissue penetration and also provide full binding specificity because the antigen-binding surface is unaltered. However, Fab and scFv are monovalent and often exhibit fast off-rates and poor retention time on the target^{ref1, ref2}. Therefore, Fab and scFv fragments have been engineered into dimeric, trimeric or tetrameric conjugates to increase functional affinity through the use of either chemical or genetic cross-links^{ref1, ref2, ref3, ref4}.
• proteolysis of intact antibodies to yield monovalent Fab fragments (e.g. in ReoPro, Remicade), however, does not easily yield molecules smaller than a Fab fragment
• single chain variable fragments (scFv) : scFv are V_L-neutral bridging peptide-V_H molecules that self-pair themselves in a Ig-like manner when assume tertiary structure. V_L and V_H sequences can be obtained through RT-PCR on > 2.5^.10⁷ hybridoma or spleen B cells by using primers specific for the variable region of each chain. The Amersham Pharmacia's Phage Antibody System (RPAS) produces a small amount of the 750 bp scFv gene, followed by in vitro ligation of randomly chosen V_H and V_L with the gene sequence coding for the linker (a neutral bridging peptide)^{ref1, ref2}. Random pairing increases donor variability in V regions and theoretically allows the creation of a library including Igs against every possible Ag (library affinity may also be improved by using particular loxP sites for chain shuffling). IgM V regions are preferred because they rarely undergo somatic hypermutation : as the latter usually positively selects simultaneous changes in both V_H and V_L regions that allow conformational stability, random pairing of such regions would often produce unstable scFvs. Ig-like molecules can be created by adding a further -C_H3 dimerization domain after V_H sequence : this allow the scFv to become immunogenic offering a T_h epitope. The fragment is then amplified using a set of restriction site primers that add Sfi I and Not I sites to its 5' and 3' ends, respectively, in preparation for enzyme digesetion and ligation into Sfi I/Not I-digested  pCANTAB 5 E phagemid for transformation of competent Escherichia coli TG1 cells.

Such mAbs can be expressed as ...
• ... membrane-bound (surface display libraries) chimaeric protein with ...
• ... protein III (pIII) from recombinant Enterobacteria phage M13, subspecies fd (phage display). The scFv cds is inserted between the leader peptide cds and the protein III NTD cds (fd-Tet^R-DOG1 phage vectors and amp^RpHEN1 phagemid vectors). The recombinant phage that are produced with the aid of M13KO7 helper phage contain a single-strand copy of the phagemic DNA encoding a specific antibody scFv gene, displayed on the phage tips as fully functional antibody scFv fusion proteins. Selection and enrichement of the desired phage is accomplished  by panning against the target antigen either on a solid phase (=> ELISA with HRP/antiM13 conjugate) or in solution using NHS-LC-biotin-labeled antigen (=> antigen/antibody complexes are then captured using streptavidin coupled to a crosslinked beaded agarose : this method allows for not only easy manipulation of antigen concentration but, based on the quantity of antigen used, selection of antibodies that exhibit high affinities or kinetics of dissociation). After washing, only the virions infected with the phage carrying the specific scFv remain attached to the plate and can be recovered through eluition at pH 2 (this phage resists such pH !). The resultant antigen-positive clone can be amplified by PCR and used to infect Escherichia coli HB2151 to produce soluble antibodies (expressed in Escherichia coli periplasm or secreted into the culture medium) for use as immunological reagents, and even further purified from other E.coli proteins by affinity chromatography and used to infect carrier Escherichia coli.
• ... protein of Saccharomyces cerevisiae
• ... secreted mAbs : by introducing an amber mutation inside the cds for transmembrane region in protein III and propagating it in a Escherichia coli strain with no suppressive mutations in tRNA for amber codon
• ... intracellular antibodies (ICAbs) / intrabody : scFv or V_H equipped with targeting signals and synthetized on cytosolic ribosomes can interact with intracellular proteins at binding affinities of 10 nM or better either to neutralize intracellular gene products or to target cellular pathways. If you have an intrabody that has an affinity close to or better than the affinity of the interacting partner, you should be able to block the interaction using the intrabody. Intrabodies offer the cell biologist the prospect of highly specific tools that can probe protein–protein interactions in situ more delicately than techniques that simply remove one of the proteins from the scene by antisense or gene knockout : because an intrabody binds to a specific site on a protein, it can block a protein's interaction with one partner while allowing it to continue acting with another. Another use is for the validation of potential drug targets, one of the first and vital steps in drug discovery : most potential target proteins are modular, so intrabodies that bind to and block the function of individual modules could help to determine which part of the protein is key to causing the disease. Placing localization signals on an intrabody allows it to be directed to various parts of the cell, such as the nucleus. scFv have been reported to fold poorly in the reducing environment of the cytoplasm and as such there has been a reluctance to use scFv-phage libraries as a source of intrabodies unless a pre-selection step to identify these rare scFvs from natural libraries or libraries of engineering scFvs that could fold properly in the absence of disulfide bonds were used. Direct phage to intrabody screening (DPIS) strategy should allow investigators to bypass much of the in vitro scFv characterization that is often not predictive of in vivo intrabody function and provide a more efficient use of large native and synthetic scFv phage libraries already in existence to identify intrabodies that are active in vivo. For example, expression of p21^rasref, erbB2^ref, huntingtin^ref and class I MHC^ref have all been individually downregulated using antibodies. Intrabodies also have important antiviral potential, particularly through their targeting of intracellular action to mandatory viral proteins such as the Vif, Tat or Rev components of HIV^ref. Antibody frameworks have been adapted that substantially improve expression levels and solubility in the intracellular reducing environment^ref. Direct in vivo selection from large libraries will greatly facilitate the isolation of many previously unknown intrabodies^{ref1, ref2}. Obviously, the expression of intrabodies in vivo can be encoded into gene therapy vectors, and this could ultimately be their most powerful clinical application.
This technology typically requires successive rounds of in vitro mutagenesis, V gene shuffling and/or panning to produce antibodies with sub-nanomolar affinities ("in vitro affinity maturation").
UK-based Cambridge Antibody Technology reduces antibody production time from months to weeks using phage display. The company houses a library of > 100 billion distinct phage antibodies. 11 human MAbs originating at Cambridge Antibody Technology have entered clinical trials, including Humira (adalimumab), the first fully human MAb approved for sale in the USA : it garnered $852 million in sales in 2004. Cambridge, Mass.-based Dyax has an automated phage-display discovery tool that allows scientists to rapidly select the best antibody, small protein, or peptide binders from libraries containing billions of candidates. Human Genome Sciences also houses it own collection of proprietary antibody targets. In 2000, the company purchased rights to technology that allows it to produce human antibodies for clinical trials without the help of an outside pharmaceutical company. The company's human MAb, LymphoStat-B (isolated at Cambridge Antibody Technology), recognizes and inhibits BLyS, typically found in high levels among patients with SLE and RA. On February 1, 2005, MorphoSys, based near Munich, Germany, announced that the first fully human therapeutic candidate based on its HuCAL technology entered clinical trials in Europe. Munich-based GPC Biotech is conducting the Phase I trial of ID09C3, an anti-MHC class II monoclonal, in patients with B-cell lymphomas. MorphoSys' HuCAL GOLD combinatorial library contains > 10 billion members; therapeutic candidates are selected using phage display. InNexus Biotechnology of Vancouver, British Columbia, has addressed that problem with its so-called SuperAntibody Technology (SAT).
InNexus' SAT platforms combine site-specific chemical conjugation and genetically engineered fused proteins to improve MAb potency, avidity, and affinity, and promote intracellular transit. Early proof-of-principle studies suggest that coupling a synthetic peptide, called a membrane-translocator sequence, to an MAb allows it to penetrate cells and target antigens from within^ref. The company is now developing SuperAntibody conjugates for the treatment of plaque associated with coronary artery disease.
Multimeric scFvs : various methods have been devised
• the most successful design was the simple reduction of scFv linker length^ref to direct the formation of
• bivalent dimers (diabodies, 60 kDa)
• trimers (triabodies, 90 kDa)
• tetramers (tetrabodies, 120 kDa).
• dimeric small immuno-protein (SIP) / minibody : V_L - V_H - dimerizing g-C_H3 or e-C_H4 domain. Spontaneously formed scFv-C_H3 dimer has shown exceptional targeting ability, particularly when the linker between the C_H3 domain and the scFv used was the IgG₁ hinge region plus a 10-amino-acid spacer.
Indeed, the first clinical trials of scFv fragments are likely to be as multivalent reagents, because they exhibit high functional affinity and have been very successful in preclinical studies^{ref1, ref2, ref3, ref4}.
Engineering multiple specificity in antibody fragments can be useful to
• bind to 2 adjacent epitopes on a single target antigen, thereby increasing the avidity
• cross-link 2 different antigens and are powerful therapeutic reagents, particularly for recruitment of CTLs for cancer treatment create bridges between a cell to be killed and a cytotoxic triggering receptor on a killer cell^{ref1, ref2}
• bispecific antibodies (BsAb) created by rodent mAbs (=> when  administered in humans elicit human anti-murine antibodies (HAMA)) contain 2 different binding specificities fused together^ref. BsAb can be produced by :
• hybrid hybridomas : fusion of 2 hybridoma cell lines into a single 'quadroma' cell line; however, this technique is complex and time-consuming, and it produces unwanted pairing of the heavy and light chains
• far more effective methods to couple 2 different Fab modules incorporate either chemical or genetic conjugation or fusion to adhesive heterodimeric domains, including designed C_H3 domains^{ref1, ref2}
• bispecific diabodies (BsDb) created by:
• covalent scFv-scFv tandems
• noncovalent association of 2 scFv consisting of the V_H and V_L domains of different specificities in an orientation preventing intramolecular pairing with the formation of a ...
• 4-domain heterodimer BsDb
• 8-domain homodimer tandem BsDb
The 2 Ag-binding domains have been shown by crystallographic analysis to be on opposite sides of the BsDb such that they are able to cross-link 2 cells. The typical plasmid operon used is as follows :



Antibody library display has superseded hybridoma technology for the selection of human antibodies through the creation of large natural and synthetic immune repertoires in vitro^{ref1, ref2, ref3}. From these libraries, specific high-affinity antibodies can be selected by linking phenotype (binding affinity) to genotype, thereby allowing simultaneous recovery of the gene encoding the selected antibody. Antibodies are usually displayed as monovalent Fab or scFv fragments and then, as required, reassembled into intact Ig or multivalent variants after selection^{ref1, ref2}. If the repertoire is sufficiently large, a high-affinity Fab or scFv can be selected directly or, more frequently, the recovered gene can be subjected to cycles of mutation and further selection to enhance affinity. Furthermore, new methods of selection and screening have been designed to specifically isolate antibodies with desired characteristics, such as enhanced stability, high expression or capacity to activate receptors^{ref1, ref2}.
• phage display : Fd phage and Fd phagemid technologies are currently the most widely used in vitro methods for the display of large repertoires and for the selection of high-affinity recombinant antibodies against a range of clinically important target molecules^{ref1, ref2, ref3, ref4}. Innovative selection methods have proved powerful for isolating antibodies against previously refractory antigens, such as new tumor-associated antigens, cell surface receptors and HLA-A1-presented peptides^{ref1, ref2}. Important improvements in selection technology have included array screening for high-avidity antibodies^ref and recovery of internalized phage from live cells to select against internalizing (human) receptors^ref. Phage technology has been applied to complete proteome analysis using membrane-based screening^ref
• libraries of mRNA-protein complexes. Ribosome display relies on stabilized complexes of antibody, ribosome and mRNA to replace bacteriophage as the display platform^{ref1, ref2, ref3}. Ribosome complexes are constructed totally in vitro, thereby eliminating the need for cell transformation and allowing the production of large libraries, 10¹⁴ members. The system is limited only by the requirement of a ribonuclease-free environment for selection and buffer compositions suitable for antibody folding. Indeed, picomolar affinity antibodies have been selected and rapid affinity maturation cycles carried out using this innovative in vitro method^{ref1, ref2}. Covalent display using puromycin-stabilized mRNA-protein complexes is an alternative strategy to ribosome display^{ref1, ref2}.
• cell surface libraries. Before the advent of bacteriophage systems, antibodies had been displayed on or in bacterial cells, although replica plating had limited screening to libraries of <10⁸. The recent development of high-speed flow cytometers has re-activated the efforts in cell surface display, and several high-affinity antibodies have been isolated by this method^{ref1, ref2}.
Bifunctional antibodies : antibodies have been fused to a vast range of molecules that provide important ancillary functions after target binding. These include :
• radionuclides
• cytotoxic drugs for cancer therapy (engineered to effectively target tumor-associated antigens at low levels and then deliver a cytotoxic payload to tumor cells
• toxins : the latest antibody-toxin conjugates are stable in vivo and minimally immunogenic^{ref1, ref2}
• peptides
• Troybodies™ (Affitech) is a novel "cellular" vaccine technology applicable to the treatment of cancer and infectious diseases. They are genetically engineered recombinant antibodies in which T-cell epitopes are inserted as loops between b-strands in Ig constant domains, and new V-regions are added which allowing the targeting of the Troybodies™ to specific APCs (such as DC or B cells) like a Trojan horse^{ref1, ref2, ref3 (full text), ref4, ref5, ref6}


Troybodies™ undergo endocytosis in APCs followed by partial breakdown of the molecules. As a result, T cell epitopes are liberated and are presented subsequently presented on MHC  molecules on the APC surface to efficiently stimulate CD4⁺ T cells. The insertion of T-cell specific epitope(s) in the C region of Ig allows the antigen binding property of the antibody to be retained. In in vitro and in vivo model systems, IgD specific Troybodies, directed against B-cells, were able to turn these APCs into 1000 times more potent stimulators of T cells as compared to unspecific delivery of the same T-cell epitope^ref.
• scFv fused to a T cell activation molecule (T body)^ref
• proteins
• cytokines (e.g. antibody-IL-2 fusion proteins^ref)
• enzymes for prodrug activation, primarily for cancer therapy^ref
• viruses for targeted gene therapy^{ref1, ref2, ref3, ref4, ref5}.
• lipids and PEGs^ref, both to enhance in vivo delivery and pharmacokinetics and to direct drug-loaded liposomes^{ref1, ref2}. As immunoliposomes, anti-transferrin receptor antibodies have been used to deliver drugs to the brain, passing through the blood-brain barrier^ref
Expression systems^{ref1, ref2} : production of recombinant antibodies for preclinical and clinical trials has been evaluated in :
• bacteria are favored for expression of small, non-glycosylated Fab and scFv fragments, usually with terminal polypeptides such as c-Myc, His or FLAG, for affinity purification^ref. Expression has been performed in bacterial systems^{ref1, ref2, ref3} and optimized for large-scale fermentations^{ref1, ref2}
• insect cells
• mammalian^{ref1, ref2, ref3} are favored for intact antibodies and, occasionally, also for expression of scFvs, diabodies and minibodies^{ref1, ref2}
• plant cells (plantibody)^ref are favored for intact antibodies and, occasionally, also for expression of scFvs, diabodies and minibodies^{ref1, ref2}. Transgenic plants show several advantages as a large-scale antibody production system: they can be grown easily and inexpensively in large quantities that can be harvested, stored and processed by using existing infrastructures. Isolation and purification of plant-made antibodies, if necessary, allow fundamental, industrial, and therapeutical applications. The maximal accumulation levels of antibodies and antibody fragments are 1-5% of the extracted proteins^ref. Currently, several biotechnological companies grow field crops to produce antibodies for ex planta applications on an industrial scale. A challenge are the implications of plant-specific glycosylation. Expression in transgenic tobacco (Nicotiana tabacum) of
• an anti-HBsAg mouse IgG₁ mAb, currently used for the industrial purification of the recombinant vaccine antigen, has been achieved. Using the sweet potato sporamin signal peptide, a KDEL (Lys-Asp-Glu-Leu) ER (endoplasmic reticulum) anchorage domain, and a heavy- and light-chain gene tandem construction, F₁ plants in which the expression of the antibody accounted for 0.5% of the total soluble proteins have been generated. The plantibody was easily purified by protein A-Sepharose chromatography with a yield of approximately 35 mg/g of fresh leaf material, and its glycosylation indicated that, irrespective of the KDEL signal, the molecule is modified in both the ER and Golgi. The plantibody compares with the ascites-derived mAb in the immunoaffinity purification of the vaccine recombinant HBsAg^ref.
• although the glycosylation pattern of plant-derived mAb (mAb^P) CO17-1A differs considerably from that of the mammalian-derived mAb (mAb^M), the biological activity of both mAbs is quite similar. mAbP heavy and light chains assembled to bind the recombinant antigen GA733-2E and specifically bound to human SW948 colorectal carcinoma cells expressing the antigen GA733-2 to the same extent as mAb^M. mAbP was as effective as mAb^M CO17-1A in inhibiting tumor growth of xenotransplanted SW948 cells in nude mice. These results suggest the promise of transgenic plants as a useful alternative way to produce full-size mAb for cancer immunotherapy^ref.
• incorporation of the N-terminal fragment of SARS-CoV S protein (S1) into tomato and low-nicotine tobacco plants genomes as well as its transcription was confirmed by PCR and RT-PCR analyses. High levels of expression of recombinant S1 protein were observed in several transgenic lines by Western blot analysis using specific antibodies. Plant-derived antigen was evaluated to induce the systemic and mucosal immune responses in mice. Mice showed significantly increased levels of SARS-CoV-specific IgA after oral ingestion of tomato fruits expressing S1 protein. Sera of mice parenterally primed with tobacco-derived S1 protein revealed the presence of SARS-CoV-specific IgG as detected by Western blot and ELISA analysis^ref.
• anti-38C13 mouse B-cell lymphoma scFv^ref
• methylotrophic yeast Pichia pastoris allows efficient production of secreted disulfide-linked, glycosylated homodimeric scFv-Fcg₁ fusion protein, albeit with high-mannose oligosaccharides. The increased size of the dimer (approximately 106 kDa vs. approximately 25 kDa for a scFv) results in a prolonged serum half-life in vivo, with t_1/2 of the b phase of clearance increasing from 3.5 h for a typical scFv to 93 h for a scFv-Fc fusion in mice. The scFv-Fc fusion is capable of mediating antibody-dependent cellular cytotoxicity against tumor target cells using human peripheral blood mononuclear cells as effectors. Finally, the Fc domain is a convenient, robust affinity handle for purification and immunochemical applications, eliminating the need for proteolytically sensitive epitope and/or affinity tags on the scFv^ref. Human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation^ref
Affinity maturation : both transgenic mice and display libraries typically produce human antibodies with binding affinities (K_D) ranging from 10^-7 to 10^-9 M. Obtaining higher-affinity antibodies is important for efficient binding to the antigenic target for in vitro diagnosis, viral neutralization, cell targeting and in vivo imaging. To improve antibody affinity, various in vitro strategies have recently been optimized to mimic the mammalian in vivo process of SHM and selection^ref. These include :
• site-specific mutagenesis based on structural information. Even with the most detailed structural information, the techniques for design of precisely complementary surfaces through interface mutations remain in their infancy. Using the cycles depicted below, affinity enhancement can be restricted to mutations in the antigen-binding surface (CDR loops).
• mutations in the underlying framework regions have frequently provided large increases in affinity, stability and expression^{ref1, ref2, ref3}.
• mutagenesis of complementarity-determining regions (CDRs)
• random mutagenesis of the entire V-domain genes can be derived from Escherichia coli mutator cells, homologous gene rearrangements or error-prone PCR
• sequential 'chain shuffling' of the 2 V genes in the Fv module is also 'random' but offers the advantage that only one V domain is altered at a time, while the other domain is kept constant to provide a defined specificity^{ref1, ref2, ref3, ref4, ref5}.
• incorporation of highly mutagenic enzymes such as mRNA reverse transcriptase and DNA polymerase with no proofreading activity to achieve a high gene mutation rate^ref. The integration of such polymerases into the ribosome display and selection process could rapidly generate large libraries of mutants.
After a decade of developing library display strategies, it is now obvious that the most successful methods rely on several cycles of mutation, display, selection (recovery) and gene amplification. These cycles of mutation and selection can be carried out using either in vitro or in vivo strategies and have been far more effective than precisely designed alterations for affinity enhancement^{ref1, ref2}.

Alternative scaffolds : intact antibodies, Fab and scFv fragments provide an antigen-binding surface comprising 6 CDR loops; these can be mutated, sequentially or collectively, to bind to a vast array of target molecules. Some target molecules are refractory to the immune repertoire, however, particularly those with cavities or clefts that require a small penetrating loop for tight binding. The natural mammalian antibody repertoire simply does not encode penetrating loops, and only rarely has this type of antibody been selected^ref. Unexpectedly, both camelids (camels, llamas and related species) and sharks produce natural, single V-like domain repertoires displaying cavity-penetrating CDR loops that complement the repertoire of conventional antibodies^{ref1, ref2, ref3}. This theory has led to a number of attempts to design single-domain display libraries in vitro, based on V domains^ref and other Ig-like scaffolds^{ref1, ref2}. These small molecules complement both antibody and peptide libraries and are expected to have improved pharmacokinetics for several clinical applications, including those that require access to buried (immunosilent) sites or clefts in enzymes, receptors and viruses^ref. Many important diagnostic targets, notably prions^ref, have also been refractory to conventional antibodies, and we expect that new molecular libraries and scaffolds will be required to provide the required binding reagents.
Biosensors and microarrays: the future of diagnosis. A likely prediction is that biosensing devices and microarrays will dominate the in vitro diagnostic market by 2005. Antibodies currently provide high-sensitivity reagents for a huge range of diagnostic kits, accounting for approximating 30% of the $20 billion per year diagnostic industry. It is therefore not surprising that antibodies are the paradigm for proof-in-principle of new biosensing devices, focused initially on glass-surface microarrays^ref. Already in 2002 we have seen more protein-friendly surfaces being developed as array platforms for antibody-based diagnosis (Triage from Biosite, San Diego, California; Protein Profiling Biochip from Zyomyx, Hayward, California; Hydrogel from Perkin-Elmer, Boston, Massachusetts). These platforms will become increasingly available over the next few years, driven by the demand for new reagents to diagnose the vast array of biomarkers stemming from proteomics discovery programs. These platforms will also be developed for robust ex vivo applications, including the detection of microbial contaminants, pesticides and biological (warfare) pathogens^ref. It is likely that we will soon witness development of display and screening technologies incorporating nanoarray robotics. By providing a highly stable, protease-resistant scaffold, engineered recombinant antibody fragments will continue to be the model for selection of high-affinity clinical targeting reagents.
Schematic representation of an intact Ig together with Fab and Fv fragments and single V (colored ovals; dots represent antigen-binding sites) and C domains (uncolored). Engineered recombinant antibodies are shown as scFv monomers, dimers (diabodies), trimers (triabodies) and tetramers (tetrabodies), with linkers represented by a black line. Minibodies are shown as two scFv modules joined by two C domains. Also shown are Fab dimers (conjugates by adhesive polypeptide or protein domains) and Fab trimers (chemically conjugated). Colors denote different specificities for the bispecific scFv dimers (diabodies) and Fab dimers and trimers.
mAb applications :
• in vitro
• chromophore-assisted light inactivation (CALI)
• fluorobodies
• catalytic antibodies / abzymes are selected from monoclonal antibodies generated by immunizing mice with haptens that are analogs of the transition state of enzyme-catalyzed reactions (e.g.  hydrolysis of carboxylic esters) behave as enzymic catalysts with the appropriate substrates. These substrates are distinguished by the structural congruence of both hydrolysis products with haptenic fragments. The haptens are potent inhibitors of this esterolytic activity, in agreement with their classification as transition state analogs^ref.
• MOPC167 abzyme binds the transition state analog p-nitrophenylphosphorylcholine with high affinity and catalyzes the hydrolysis of the corresponding carbonate 1. MOPC167 catalysis displays saturation kinetics with catalytic constant (kcat) = 0.4 min-1 and Michaelis constant (K_M) = 208 mM, showed substrate specificity, and was inhibited by p-nitrophenylphosphorylcholine. The rate of the reaction was first order in hydroxide ion concentration between pH 6.0 and 8.0. The lower limit for the rate of acceleration of hydrolysis by the antibody above the uncatalyzed reaction was 770^ref.
• 28B4 abzyme catalyzes periodate oxidation of p-nitrotoluene-methyl sulfide to sulfoxide (mice were immunized with an aminophosphonic acid hapten) : K_d = 52 nM and k₃/K_M = 190,000 M^-1s^-1ref
• in vivo
• immunoprophylaxis
• immunotherapy
Web resources :
• Antibody Engineering Group
• Antikörper Engineering - Protein Design by Prof. Dr. Arne Skerra
• Human recombinant antibodies and phage display by Dr.Jos Raats, Department of Biochemistry, University of Nijmegen, The Netherlands
• Humanization by design by José Saldanha
• Information about phage display technology
• Phage display information
• Slideshows by Andreas Plückthun lab
• The Antibody Engineering Page by Roland Konterman
• The Recombinant Antibody Page by Stefan Dübel
• Therapeutic Ab Human Homology Project (TAHHP) by Mike Clark
Web resources :
• cloning
• Cosmid packaging protocol
• Cosmid fingerprinting
• Codes for cosmid vectors
• Single stranded template from pBluescript phagemid
• l DNA Miniprep
• Largescale M13 prep
• M13 PEG prepI
• M13 PEG prepII
• M13 helperphage
• M13 ThermoMAX prep
• M13 RF prep largescale
• Preparation of M13 sublibraries
• PCR of M13 inserts
• Easy YAC Preparation Method
• Gene library at COPE
• recombinant DNA
• Muteins at COPE
• Cytokine assays
• Cytokine fusion toxins
• Recombinant cytokines
• introducing sequence-specific modifications in another nucleic acid molecule : it allows to maintain the appropriate control of gene expression because the endogenous promoter is utilised.
• at DNA level, by homologous recombination (HR) : low efficiency
• small DNA fragments (SDF) / single-stranded oligodeoxynucleotides (ssODN) are 400-800 bp long homologous to the sequence of the mutant gene except that for a centrally located mismatch that code for the normal, rather than the mutant sequence => small fragment homologous replacement / recombination (SFHR)
• triplex forming oligonucleotides (TFO) thanks to Hogsteen base pairings (G in oligonucleotide binds to G in duplex ; T in oligonucleotide binds to A in duplex). The forming of a stable triple helix competes with the binding of transcriptional factors in the promoter region. When TFO are covalently linked to a DNA-crosslinking agent (psoralen), the induced mutagenesis seems to be associated with NER or transcription-coupled repair pathways.
• RNA/DNA hybrid oligonucleotides (RDO) / chimeraplasts : there is a significant increase in pairing efficiency between an oligonucleotide < 50 bases and a genomic DNA target if RNA replaces DNA in a portion of the targeting oligonucleotide. The original chimeric 68-mer design incorporated 10 2'-O-methylated RNA residues flanking each side of a 5 bp stretch of DNA, poly-T hairpin loops, a 3' GC clamp and a complementary all-DNA strand resulting in a stable, nuclease-resistant duplex molecule. It is postulated that the "mismatched" chimeraplast complexes with the genomic DNA and creates the illusion of a genetic mutation leading to the recruitment of endogenous repair functions.
Efficiency can be improved by covalently binding a TFO to a linker oligonucleotide, which in turn non covalently bind and drives donor DNA (DD) (i.e. ODN, SDF or RDO) to the proper site, increasing local concentration and provoking recruitment of proteins involved in homologous pairing => guided homologous recombination (GOREC)
• at RNA level
• ribozymes (group I introns) that catalyze RNA trans splicing between wild-type and mutated mRNAs.
• spliceosome-mediated trans-splicing (SMaRT) : cells are transfected with very high titres of wild-type gene producing the so-called pre-therapeutic RNA molecule (PTM), which is designed to promote RNA trans-splicing with the endogenous mutated RNA.
• RNA editing : antisense oligoribonucleotide + ADA for dsRNA
• inhibiting the expression of a gene ("antigene" strategies)
• at DNA level
• triplex forming oligonucleotides (TFO) : thanks to Hogsteen base pairings (G in oligonucleotide binds to G in duplex ; T in oligonucleotide binds to A in duplex), the forming of a stable triple helix in the promoter region competes with the binding of transcriptional factors
• double-stranded oligodeoxynucleotides (dsODN) corresponding to the promoter sequence act as decoys
• unmodified dsODN : relatively easy degradation by nucleases prevalent in sera and cells.
• ab-anomeric dsODN
• modified linkages : insensitivity to RNase H, mutational potential by hydrolyzed modified nucleotides, lack of sequence-specific binding effects of ODN-based gene therapy and immune activation.
• methylphosphonate dsODN
• phosphorothioate dsODN
• circular dumbbell (CD) dsODN constructed by the enzymatic ligation of the 3' and 5' ends of dsODN exhibit increased stability to exonucleases, easy uptake and a nontoxic unmodified backbone, which resembles natural DNA.
• peptide nucleic acids (PNA) are nucleic acid in which the phosphodiester units of the backbone are replaced by N-(2-aminoethyl)-Gly units. The various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. PNAs are depicted like peptides, with the N-terminus at the first (left) position and the C-terminus at the right. Since the backbone of PNA contains no charged phosphate groups, the binding between PNA/DNA strands is stronger than between DNA/DNA strands due to the lack of electrostatic repulsion. PNA/PNA binding is stronger than PNA/DNA binding. Due to their higher binding strength it is not necessary to design long PNA oligomers for use in these roles, which usually require oligonucleotide probes of 20-25 bases. The main concern of the length of the PNA-oligomers is to guarantee the specificity. PNAs are not easily recognized by either nucleases or proteases, making them resistant to enzyme degradation. PNAs are also stable over a wide pH range. Finally, their uncharged nature should make crossing through cell membranes easier, which may improve their theraputic value. It has been hypothesized that the earliest life on Earth may have used PNA as a genetic material due to its extreme robustness, and later transitioned to a DNA/RNA-based system
• gripNA are 18-mer probes that possess a negatively charged backbone that increases solubility preventing in vivo aggregation (an obstacle in using PNAs). But the presence of a single base mutation either prevents or destabilizes duplex function.
• at RNA level (RNA silencing / antisense agents )
• RNA promoter decoys for RNA viruses (e.g. TAR- and RRE-decoys for HIV-1)
• RNA interference (RNAi)
• antisense ssRNAs (asRNAs), provided that these find a corresponding target mRNA that they can immediately hop onto. Furthermore it forms duplexes with cell RNAs that cannot be translated
• injection
• overexpression of antisense ssRNA from a transgene
• dsRNAs switch genes off up to 10 times more quickly than their single-stranded counterparts.
• feed dsRNA
• soak in medium containing dsRNA
• inject dsRNA
• transfect dsRNA
• overexpress dsRNA or a dsRNA hairpin from a transgene corresponding to the sequence of a gene's promter (in plants). Such transcriptional silencing is accompanied by (and perhaps mediated by) methylation of the DNA sequences in the promoter region of the silenced gene. The gene is silenced because it is no longer transcribed, unlike RNAi or PTGS, in which the mRNA is transcribed at normal levels but then destroyed. Even in such promoter-based transcriptional silencing, the dsRNA is converted to siRNA-like small RNAs. Determining whether these siRNAs are part of the transcriptional silencing pathway or merely reflect the nonproductive entry of a bit of the dsRNA into the RNAi pathway is unknown.
• injection of short / small hairpin RNAs (shRNAs) (synthetic molecules modelled on siRNA with a hairpin secondary structure) are more effective
• DNA-directed RNA interference (ddRNAi) : a DNA construct is introduced into a cell, leading to the production of a double-stranded RNA that is cleaved into siRNA. Unlike traditional RNAi, ddRNAi does not provoke an interferon response in cells.
Problems :
• RNA is a million times more fragile than DNA. In solution, it breaks apart in minutes, so researchers need to find ways to stabilize the molecule for long enough for it to take effect in the human body
• fathoming the best combination of constituent chemical letters to treat specific diseases, to make sure that the RNA does not act off target and damage healthy cells
• to ensure that RNA reaches the right part of the right cell in the right part of the body
• unlike previous nucleic acid therapeutics, siRNAs have the potential to elicit immune responses via interactions with TLR3 and trigger IFN responses like long, dsRNA and its analogs, such as poly(I:C). Recently, the safety of siRNAs has been questioned because they have been shown to trigger an IFN response in cultured cells. Anyway it is possible to administer naked, synthetic siRNAs to mice and downregulate an endogenous or exogenous target without inducing an interferon response^ref. Davis and colleagues gave mice naked synthetic siRNAs against fatty acid synthase (FAS), c-MYC, or luciferase, either through intraperitoneal injections or via the tail vein using either low-pressure (1% v/w) or high-pressure (10% v/w) methods. Different injection methods can lead to different concentrations of material in various organs. siRNA fails to trigger a strong type I IFN-a response regardless of which siRNA was injected or the injection method. In comparison, injections of poly(I:C) trigger strong interferon responses. While poly(I:C) triggers a strong IL-12 response, the siRNAs does not. To confirm that siRNAs reached an intracellular target and functioned in a sequence-specific manner, the researchers injected mice with plasmids containing the luciferase gene and siRNA against either luciferase or an unrelated sequence. Live whole-animal imaging showed that mice co-injected with the luciferase-targeting siRNA displayed a significant downregulation of luciferase expression in the liver that was not observed with the control siRNA. High-pressure tail vein injection of siRNA against FAS also reduced the level of FAS mRNA in mouse liver. This downregulation was not seen with low-pressure tail vein injection of the same siRNA at the same dose, consistent with prior observations that high pressure is required to downregulate a gene with siRNA in the livers of mice, even if the siRNA is chemically stabilized. But one still has the issue of how much of a response they could have measured. For many people applying siRNA strategies to use in patients, even small interferon responses could become a major issue, as are off-target results, when seeking approval from regulatory agencies. One has to be careful how detectable they are. To reconcile their results with prior cell culture findings, Davis and colleagues suggested investigating any influence that cell type, intracellular trafficking, siRNA concentration, method of siRNA preparation, and siRNA sequence had on immune response. For instance, it is moving from naked nucleic acids to non-viral delivery systems. Past studies in cultured cells found interferon response from expressed siRNAs using lipid vectors. It may be that lipid release delivery in itself causes some sort of stress reaction, and it could turn out some sequences have some motif in them that might turn out to be immunogenic. Lipid-delivered siRNAs are potent inducers of IFN-a and type I IFN gene expression, whereas the same sequences when expressed endogenously are nonimmunostimulatory^ref.
Designing the perfect siRNA : choosing siRNAs in an empirical process, as the rules that govern efficient siRNA-directed silencing are still unknown. On the basis of the analyses of a small number of target genes, several groups have proposed a set of guidelines to seek to narrow the choices of siRNAs that could potentially silence gene expression^{ref1, ref2}. Several sequence motifs are consistent with effective siRNA-directed silencing, including AAN₁₉TT, NAN₁₉NN, NARN₁₇YNN and NANN₁₇YNN (where N is any nucleotide, R is a purine and Y is a pyrimidine). When choosing siRNAs, regions of complementary DNA are selected that have non-repetitive sequences. Intronic sequences are avoided as mammalian RNAi is a cytoplasmic process^ref. Some groups suggest choosing siRNAs with 50% GC content (30-70%). Observations indicate that sequences with an even representation of all nucleotides on the antisense strand are favoured and that regions with stretches of a single nucleotide, especially G, should be avoided. Some have suggested that the use of 2'-deoxythymidines for the 2-nt 3' overhangs might protect siRNAs from exonuclease activity^ref. However, many groups have found that siRNAs that have ribonucleotides in te overhangs shown no obvious impariment in silencing activity when compared with the same siRNA sequence with 2'-deoxythymidine overhangs. There are several other parameters, in addition to the sequence considerations, that might affect the efficiency of siRNA-directed mRNA cleavage. Any region of mRNA can be targeted, however, sequences that are known sites for mRNA-binding proteins in the 5' UTR, 3'UTR, start codon or exon-exon boundaries should be avoided. Although some suggest selecting sequences that are 50-100 nt downstream of the start codon^ref, other observations indicate that there is a predilection for effective siRNA-directed silencing  towards the 3' portion of the gene. The choice of siRNA is dictated by the sequence of the target gene, and sometimes, siRNAs must be chosen that do not have many of the parameters for efficient gene silencing. These potential parameters require systematic testing before they are codified into a set of rules that unequivocaly promote efficient target-gene silencing. As these rules have not been tested systematically, researchers seeking to silence expression should synthesize several siRNAs to a gene and validate the efficiency of each. To ensure that the chosen siRNA sequence targets a single gene, a BLAST search of the elected sequence should be carried out against sequence databases such as EST or Unigene libraries using the NCBI website. Sequences in these databases that share partial homology to siRNAs might be targeted for silencing by the siRNA. Potential off-target effects of the siRNA might be minimized by choosing an siRNA with maximum sequence divergence from the list of genes with partial sequence identity to the intended mRNA target. The efficiency and specificity of RNAi depend on the position and sequence of the siRNA used. So how do you pick out the right 21-nucleotide sequence to knock down your favorite gene? Academic groups and companies have developed design tools, available on the Web, to help with the process. Short interfering RNAs are duplexes composed 21-nucleotide sense antisense strands, paired in a manner to have a 2-nucleotide 3' overhang. The sense strand does not contribute to the silencing effect (although it can contribute to off-target effects), which means that, for effective gene silencing, the antisense strand has to be incorporated into the RNA-induced silencing complex (RISC). It turns out that the relatively low thermodynamic stability in the 5' end of the antisense siRNA strand, as compared with a higher thermodynamic stability in the 5' end of the sense strand, leads to a bias for the incorporation of the antisense strand into the RISC. Once the antisense siRNA strand goes into the RISC, there are some attributes of the siRNA sequence that correlate with efficiency. Dharmacon's Anastasia Khvorova identified 8 such factors, such as low CG content and lack of inverted repeats^ref. More recent work by Zamore and colleagues showed that 5' end of siRNA has largest effect on binding target of mRNA^ref. The region more toward the 3' end needs to be paired with mRNA for the message to be cleaved, but it is not critical for binding. Web-based design tools incorporate these 'rules' to select 21-nucleotide sequences with the right attributes, and many will even BLAST search the NCBI database against EST libraries to ensure that the chosen sequences target only one gene. The bioinformatics group at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts has developed the siRNA Selection Web Server. Users provide the accession number of a gene or a sequence they want to target and then either select a predefined siRNA sequence pattern or opt for their own pattern. Several filters can be applied to the selection process, such as GC %, base variations and number of repetitive bases. The program produces several candidate siRNA sequences ranked by the degree of specificity. The site is available free to academic researchers, but requires user registration to limit the number of searcher per individual investigator to 15 per day. Updates to the site will permit the option of designing longer siRNAs and provide assistance in predicting shRNAs. Another free site for academic users is siSearch. The RNAi OligoRetriever site designs DNA oligonucleotides that can be used to synthesize siRNAs by in vitro transcription with T7 DNA polymerase or as shRNAs transcribed from vectors containing the human U6 promoter. Many companies that provide RNAi reagents also offer Web design tools free of charge. However, the public resources available on Dharmacon's and Ambion's websites, for example, use algorithms that are less advanced than the ones these companies use for their in-house design of siRNAs. MWG Biosciences' website offer a mechanism to convert siRNA sequences to create plasmid vectors expressing hairpin duplex RNA. In addition, many websites offer information about how to make siRNAs and how to use them. The siRNA User Guide,  is a popular resource. Ambion's website also provides a wealth of articles and protocols for RNAi methodologies. Dharmacon will soon be publishing a book, RNA Interference: Technical Reference & Application Guide, that can be ordered from the company's website.
Potential determinants of efficient siRNA-directed gene silencing : sequence determinants intrinsic to the siRNA, the mRNA or both might affect the efficiency of each step of the siRNA-directed mRNA cleavage that results in efficient gene silencing
• siRNA :
• incorporation into the RISC and stability in RISC
• basepairing with mRNA
• cleavage of mRNA
• turnover of mRNA after cleavage
• mRNA
• the position of the siRNA-binding target region
• secondary and tertiary structures in mRNA
• binding of mRNA-associated proteins
• basepairing with siRNA
• the rate of mRNA translation
• the number of polysomes that are associated with translating mRNA
• the abundance and half-life of mRNA
• the subcellular location of mRNA
Limitations of gene silencig by transfected siRNA : although siRNAs have proven to be very potent inhibitors of gene expression and have allowed the elucidation and better understandin gof gene functions in many different cell lines and organisms, there are several limitations to siRNA-knockdown technology.
• transient nature of the response : the transduction of siRNA into cells leads to only a transient knockdown of the gene of interest. As siRNAs seem to be relatively resistant to degradation, the transient nature of the knockdown is determined by the rate of cell growth and the dilution of the siRNAs below a crucial threshold level that is necessary to maintain the inhibition of gene expression. In actively dividing cells, the duration of silencing is directly related to the number of cell doublings. For example, in HeLa cells, which double approximately every 24 hours, the maximum amount of silencing is usually seen 72 hours post-transfection, depending on the gene targeted^ref. However, the knockdown of a gene leads to a decrease in the doubling time. In these cells the maximum level of silencing was observed at 96 hours and the length of the silencing was extended by several days. Another factor that could limit siRNA-mediated silencing is the half-life of the protein. It might be difficult to effectively silence genes that encode proteins with long half-lives by transient transfection of siRNA
• transduction problems : the introduction of siRNAs to mammalian cells has been accomplished by the transfection of the siRNAs using lipid-based reagents^{ref1, ref2}. Each cell type must be oiptimezed with respect to the number of cells plated and the cells:siRNA:lipid-carrier ratio for efficient transfection. There are many cell lines that are refractory to transfection includin gmany primary cells, which might require electroporation for the delivery of siRNAs^{ref1, ref2}. Although this technique increases the number of cells that have taken up siRNAs, many cells die during electroporation.
• non-renewable nature of siRNAs : unlike plasmid DNA, which can be grown in bacteria for the production of large amounts of plasmid DNA vectors, siRNAs must be chemically or enzymatically synthesized, which remains a costly process
Designing shRNA-expressing vectors : in general, chemically synthesized siRNA sequences that are effective at silencing gene expression are also effective when generated from short hairpin shRNAs. However, the length of the stem and the size and composition of the loop might be important for the efficiecny of silencing. Stem lengths of 19-29 nucleotides have been shown to silence genes effectively^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}, which indicates that stem length is not the main parameter governing effective target-gene silencing. Loops that vary from 4-23 nt have been described^{ref1, ref2,ref3, ref4, ref5, ref6, ref7}, which indicates that loop lengths are also not the main parameter governing efficient gene silencing. In a direct comparison of 5-, 7- and 9-nt loops using a constant 19-nt duplex, the 9-nt loop (5'-UUCAAGAGA-3')^ref was the most efficient silencer. It should be noted that the 9-nt loop might actually form a 5-nt loop because of U:A and U:G base pairs at the ends. As 21-22-nt short RNA were generated from a 19-nt duplexed region, processing of the 19-nt stem would require Dicer cleavage in the loop sequence^ref. In this case, the sequence and potentially the length of the loop might be more crucial for processing  In constructs that have a longer stem, Dicer could choose numerous cleavage sites without having to cleave in the loop. So, choosing hairpin structures with duplexed regions that are longer than 21 nt, regardless of loop sequences and length, might promote the most effective siRNA-directed silencing. More experiments are needed to establish the contribution of the stem and loop to the effectiveness of Dicer processing and to gene silencing. There is increasing evidence that long regions of SSRNA 5' and 3' of the hairpin RNA affect the ability to target mRNA cleavage^{ref1, ref2, ref3}. It ssems that shorter duplex RNAs are more sensitive to the surrounding RNA sequence that longer duplex RNAs. The incorporation of a 70-nt pre-miR30 micro (mi)RNA sequence in a larger transcript was processed and silenced gene expression, presumably because it was not procesed by Dicer^ref. Others produced similar results with a RNA-polymerase-II-driven shRNA expression construct^ref. A U6 expression cassette containing the first 27 nt of the endogenous transcript had no detrimental effect on gene silencing^ref. However, unlike a random sequence, the first 27 nt of the U6 transcript encodes a stable hairpin structure, which might not inhibit, but actually augment production of the short RNA, thereby increasing Dicer processing near the hairpin construct.
Comparison of plasmid-based versus siRNA silencing : there are 2 principal advantages of siRNA transfection over plasmid-based gene silencing. First, siRNA transfection is more efficient than plasmid DNA transfection. More cells will silence gene expression after siRNA transfection. Second, the initiation of siRNA-transfected silencing is immediate. Plasmid-based strategies require transcription and in the case of hairpin RNA, Dicer processing. There are 2 principal advantages of plasmid-based RNAi expression systems over siRNA transfection. First , plasmid DNA can be readilly regenerated. Second, the duration of silencing can be extended. Transfection of siRNAs leads to transient silencing and might not work for genes that encode proteins with long half-lives. Cell lines can be created that stably express the short hairpin (sh)RNA and a drug-resistance marker (either on the same plasmid or from a co-transfected plasmid). Stably silenced clones can be maintained indefinitely. After plasmid transfection and drug selection for cell expressing the resistance marker, populations of cells are derived that have heterogeneous levels of silencing. To derive a homogeneous population of cells that can efficiently silence gene expression, single-cell clones must be obtained and screened, which can be a laborious process. However, the utility of plasmids will be limited in cell lines that are difficult to transfect and that cannot be grown for long periods of time in culture, such as primary cells.
Web resources :
• Ambion's siRNA target finder and design tool
• The Hannon's lab siRNA selection site
• The Tuschl lab's siRNA user guide
• Jack Lin's siRNA sequence finder
• companies offering siRNA design and delivery products : Allele Biotechnology, Alnylam, Amaxa Biosystems, Ambion, Inc., BD Biosciences, B-Bridge International, Inc., BioCat, BioChain, Bioneer, Bio-Rad Laboratories, BioVision, Boca Scientific, Cell Signaling Technology, Cenix Bioscience, ChemGenes,  Corporation, Clontech, Cyto Pulse Sciences, Inc., Dharmacon, Epicentre, Eurogentec, Galapagos Genomics, Gene Link, Genlantis, Glen Research, MRC Geneservice, GenScript, Genospectra, Gene Therapy Systems Inc., Genoway, Imgenex Corp., Integrated DNA Technologies, Invitrogen, InvivoGen, Molecula, Mirus, MWG Biotech, Nature Technology Corporation, New England Biolabs, Novagen (EMD Biosciences), OligoEngine, Open Biosystems, OrbiGen, OZ Biosciences, Panoramics, Pierce Chemical, Polyplus Transfection, Promega, Proligo, Protiva, Qiagen, RNAx, Roche Applied Science, Sigma Aldrich, Sirna Therapeutics, Spring Bioscience, Stratagene, SuperArray Bioscience Corporation, System Biosciences, Tebu-Bio, Thermo Electron Corporation, Upstate
• (non-ssRNA) antisense technology : sometimes cell RNA is not available for duplex formation due to its tertiary structure
• oligodeoxynucleotides (ODN) : they are preferred to antisense RNA because of better nuclease resistance and induction of RNAse H activity.
• phosphorothioate oligodeoxynucleotides (PT-ODN) have still greates nuclease resistance
• peptide nucleic acids (PNA)
• deoxyribozyme / DNAzymes^ref : the 33-mer ('10-23') ssDNA enzyme is comprised of a catalytic domain of 15 deoxynucleotides, flanked by 2 substrate-recognition domains of 7-8 deoxynucleotides each. The RNA substrate is bound through Watson-Crick base pairing and is cleaved at a particular purine-pyrimidine (RY) phosphodiester (AU = GU >> or = GC >> AC : substitution of deoxyguanine with deoxyinosine such that the base pair interaction with the RNA substrates core C is reduced from 3 hydrogen bonds to 3 enhances cleavage rate by up to 200-fold^ref) located between an unpaired purine and a paired pyrimidine residue. Despite its small size, the DNA enzyme has a catalytic efficiency (k_cat/K_m) of approximately 10⁹ M^-1.min^-1 under multiple turnover conditions, exceeding that of any other known nucleic acid enzyme. By changing the sequence of the substrate-recognition domains, the DNA enzyme can be made to target different RNA substrates. For applications in vivo, they have to be stabilised against nucleolytic attack by the introduction of modified nucleotides (3'-3'-inverted thymidine, phosphorothioate linkages, 2'-O-methyl RNA and locked nucleic acids (LNAzyme : an RNA mimic, fitting seamlessly into an A-type duplex geometry)) without obstructing cleavage activity. DNAzymes conjugated with nuclear export signal (NES) peptide was shown to be taken up and localized in cytoplasm^ref. Photolyase DNAzymes can catalyze photochemistry, e.g. cofactor (serotonin)-dependent and -independent photorepair of thymine dimers^ref. Mg²⁺ ion is essential to form the active binary complex between the catalytic DNA and the substrate, and heat-treatment is effective to prevent formation of the inactive quaternary complex between the 2 enzymes and the 2 substrates^ref. Current methods for their delivery in vivo include electroporation^ref . Deoxyribozymes with 2'-5' RNA ligase activity should be particularly useful for preparing site-specifically modified RNAs for studies of RNA structure, folding, and catalysis^ref.
• ribozymes : they cleave RNA molecules in a sequence-specific manner rendering them untranslatable and prone to rapid degradation by intracellular nucleases : recognition sequence can be modified ad hoc. The delivery of DNA-based ribozyme expression constructs by viral or non-viral vectors has been widely used in vitro and in vivo but this approach is hindered by problems with cell specificity, efficacy or safety issue, expecially in vivo. The alternative approach, exogenous ribozyme drug delivery, offers a direct way of ribozyme application which is not based on gene therapy : this requires, however, extensive chemical modifications of all-RNA ribozyme molecules due to their high instability in biological sera and their poor cellular uptake. They can be protected by complexation with a low molecular weight polyethylenimine (LMW-PEI).
• inhibiting the function of a protein through noncovalent interactions
• oligonucleotides that specifically bind and inhibit the protein (adaptamer or aptamer) [in case of intoxication the antidote is just the complementary sequence !]

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