WO2009071722A1 - PROCÉDÉS ET TROUSSES POUR LA PRÉPARATION DE GÉNOTHÈQUES D'ARNsi SPÉCIFIQUES D'UN TRANSCRIPTOME PAR TRANSCRIPTION CONVERGENTE - Google Patents

PROCÉDÉS ET TROUSSES POUR LA PRÉPARATION DE GÉNOTHÈQUES D'ARNsi SPÉCIFIQUES D'UN TRANSCRIPTOME PAR TRANSCRIPTION CONVERGENTE Download PDF

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WO2009071722A1
WO2009071722A1 PCT/ES2008/000758 ES2008000758W WO2009071722A1 WO 2009071722 A1 WO2009071722 A1 WO 2009071722A1 ES 2008000758 W ES2008000758 W ES 2008000758W WO 2009071722 A1 WO2009071722 A1 WO 2009071722A1
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vector
sequence
population
promoters
oligonucleotides
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PCT/ES2008/000758
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Spanish (es)
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José Antonio Pintor Toro
Miguel Angel Moreno Mateos
Ivan Valle Rosado
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Newbiotechnic, S.A.
Consejo Superior De Investigaciones Científicas
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Publication of WO2009071722A1 publication Critical patent/WO2009071722A1/fr

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised
    • C12N2330/31—Libraries, arrays

Definitions

  • the invention relates to methods for obtaining libraries of siRNAs from a population of polynucleotides that corresponds to a transcriptome associated with a specific biological process where the RNA chains that form the siRNA result from the transcription of the sense chain and antisense of a double stranded DNA by the action of two convergent promoters.
  • the invention provides kits for the realization of said methods as well as methods for the identification of genes relevant to a certain biological process in which the libraries of the invention are used.
  • RNA interference is a very useful tool to reduce the expression of a gene of interest.
  • siRNAs small interfering RNAs
  • the siRNAs are incorporated together with the complementary sequence mRNAs in a multiproteic complex called RISC, where degradation of the mRNA takes place by means of the Dicer protein.
  • siRNAs that contain the sequence that gives rise to the siRNAs under the control of a promoter, of The synthesis of the siRNAs takes place inside the cell after the insertion of the vectors that encode the siRNAs into the cell.
  • One type of vector used for the expression of intracellular siRNAs is one in which the two regions of DNA encoding the two chains of the siRNA are arranged in tandem in the same DNA chain separated by a separating region that, when transcribed, forms A loop.
  • RNA molecules thus formed are digested by Dicer, an RNase III enzyme, giving rise to mature siRNAs (Hammond, SM et al., Nature, 2000, 404: 293-296).
  • a single promoter directs the transcription of the DNA molecule that gives rise to shRNA.
  • Promoters suitable for use in these vectors include RNA polymerase III (pol III) type III promoters, in particular, U6 and Hl promoters. Examples of these vectors have been described in Brummelkamp, TR et al., (Science, 2002, 296: 550-553), Paddison, PJ.
  • each of the chains that form the siRNA is formed from the transciption of a different transcriptional unit. These vectors are in turn divided into divergent and convergent transcription vectors.
  • divergent transcription vectors the transcriptional units encoding each of the DNA chains that form the siRNA are located in tandem in a vector so that the transcription of each DNA chain depends on its own promoter, which can be same or different (Wang, J. et al., 2003, Proc.Natl.Acad.Sci.USA., 100: 5103-5106 and Lee, NS, et al., 2002, Nat.Biotechnol., 20: 500- 505).
  • the DNA regions that give rise to the siRNA are centered forming the sense and antisense chains of a DNA region that is flanked by two inverted promoters. After transcription of the sense and antisense RNA chains, these they will form the hybrid corresponding to the functional siRNA.
  • Vectors have been described with inverted promoter systems in which 2 U6 promoters are used (Tran, N.
  • siRNAs or vectors capable of generating the siRNA of a specific gene For the inactivation of a gene of interest it is necessary to have siRNAs or vectors capable of generating the siRNA of a specific gene.
  • a problem associated with mRNA inhibition methods is that, for unknown reasons, only 25% of the siRNAs designed according to the sequence of the mRNA targets are capable of inducing the degradation of said mRNAs. Therefore, obtaining a specific functional siRNA requires the synthesis and analysis of multiple siRNA until one is found that has the desired activity.
  • siRNA library specific to the luciferase gene based on a vector in which the RNA chains that form the siRNA are transcribed from an Hl promoter and a U6 promoter organized in a convergent and inverted way.
  • This library has been obtained by inserting in the aforementioned vector of sequences derived from the luciferase gene selected, by an algorithm, by its theoretical suitability for use as a siRNA and in which three nucleotides in the central region of the siRNA are random, so that the library would comprise 64 different siRNAs.
  • the siRNAs libraries serve not only to identify siRNAs capable of blocking the expression of a known target gene, but also to identify genes that are involved in a given process. For this, the cells are transfected with a library of siRNAs, the biological process of interest is analyzed, the cells in which the process is altered are identified and finally the sequences of the siRNAs present in said cells are characterized. Those sequences that have a high similarity with the sequences of one or more of the siRNAs that show this effect are candidates to be involved in the biological process analyzed. An example of this type of scrutiny is found in Moffat. et al.
  • siRNA libraries as well as methods for their synthesis that allow obtaining random siRNAs, without going through a shRNA intermediate, and that represent the population of all possible specific siRNAs of the mRNAs involved differentially in a given biological process or mechanism.
  • the invention relates to a method for the preparation of a vector library that encodes a population of specific siRNAs of a transcriptome associated with a biological process comprising:
  • step (c) convert the oligonucleotides obtained in step (b) into double chain oligonucleotides
  • step (d) inserting the double chain oligonucleotides obtained in step (c) into a cloning vector comprising two independent promoters that act in a convergent manner and wherein the oligonucleotide is inserted between both promoters so that one of the promoters controls the Transcription of a DNA strand of the oligonucleotide and the other promoter controls the transcription of the complementary strand.
  • the invention relates to a method for the preparation of a vector library capable of generating a population of specific siRNAs of a transcriptome associated with a biological process comprising:
  • step (a) fragment a specific cDNA population of a transcriptome; (b) modify the fragments obtained in step (a) to generate protruding and cohesive ends
  • step (c) insert the fragments obtained in step (c) into a linearized cloning vector containing cohesive ends compatible with the ends of the oligonucleotides introduced in step (b) wherein said vector comprises two independent promoters that act in a convergent manner and wherein the oligonucleotide is inserted between both promoters so that one of the promoters controls the transcription of a DNA strand of the fragment and the other promoter controls the transcription of the complementary strand.
  • the invention relates to a library obtainable by a method such as defined in the first and second aspects of the invention.
  • the invention relates to a method for obtaining vectors for the expression of a siRNA capable of interfering in a biological process comprising the steps of
  • step (c) rescue the vector or population of vectors from the library from the cells or organisms identified in step (b).
  • the invention relates to a method for the identification of genes involved in a given biological process comprising:
  • the invention relates to a kit for the preparation of a library of the invention comprising
  • each oligonucleotide comprises a central region of random sequence flanked by regions of constant sequence
  • a cloning vector comprising one or more restriction targets that are flanked by two independent promoters that act in a convergent manner.
  • the invention relates to a kit for the preparation of a library of the invention comprising
  • a cloning vector comprising one or more restriction targets that are flanked by two independent promoters that act convergently.
  • Figure 1 depicts the scheme of the restriction enzyme cut BbvII in the random oligonucleotide.
  • the underlined sequence corresponds to the BbvII recognition site.
  • the doubly underlined sequence corresponds to the transcriptional terminator.
  • Figure 2 represents the scheme of the process of formation of the double chain siRNA under the control of a double promoter system by convergent transcription.
  • Figure 3 shows the scheme of the process of selecting molecules that correspond to specific mRNA sequences present in the cell exposed to nocodazole.
  • Figure 4 depicts the process of isolation and purification of the siRNAs by elution and amplification of the oligonucleotides using primers complementary to the constant ends of the oligonucleotides.
  • the authors of the present invention have shown that it is possible to generate libraries of random siRNAs using vectors in which the transcription of the siRNA chains takes place from two convergent promoters, that is, in which the siRNAs are produced directly if you need to go through a shRNA intermediate. These libraries have greater efficiency and allow the identification of genes involved in a certain biological process. Likewise, it has been observed that the identification of siRNAs involved in a certain biological process is possible if the oligonucleotides that are inserted into the vector and that serve as a template for the synthesis of siRNAs have been previously enriched from a population of cDNA mRNA derivatives that are expressed mostly in cells in which the process to be analyzed is manifested.
  • the pre-selection stage significantly increases the probability of obtaining siRNAs with different interference capacity (which is critical in certain processes since the loss of total expression of phenotypes other than partial loss), and allows obtaining specific libraries of each transcriptome associated with a specific biological process, since siRNAs will target the genes expressed mostly under specific conditions.
  • the invention relates to a method for obtaining a library of specific siRNAs of a transcriptome associated with a biological process (hereinafter the first method of the invention) comprising: (a) contacting a population of specific cDNAs of a transcriptome associated with a biological process with a population of oligonucleotides under conditions that allow hybridization between the cDNA chains and oligonucleotide chains, wherein the oligonucleotides comprise a central region of random sequence flanked by regions of constant sequence; (b) select those oligonucleotides from the population that hybridize specifically with said cDNA population, (c) convert the oligonucleotides obtained in step (b) into double chain oligonucleotides and
  • step (d) inserting the double chain oligonucleotides obtained in step (c) into a cloning vector containing two independent promoters that act in a convergent manner and wherein the oligonucleotide is inserted between both promoters so that one of the promoters controls the transcription of an oligonucleotide DNA chain and the other promoter controls the transcription of the complementary chain.
  • Transcriptome associated with a specific biological process means, in the context of the present invention, the set of mRNA that is expressed in a given cell as a result of the activation of a certain biological process and that is absent or present in minor to a lesser extent) in the cell prior to the activation of said process.
  • the preparation of the specific cDNA population of a transcriptome associated with a given biological process can be carried out using any of the methods widely known to the person skilled in the art and which are described in Chapter 25, section B in Ausubel, FM et al. (Current Protocols in Molecular Biology, John Wiley and sons, inc, eds, Ringbou edition, December 2004). Suitable methods for the preparation of said cDNA population include
  • a specific cDNA population for a transcriptome associated with a given biological process is available, it is contacted with a population of single-chain oligonucleotides of partially random sequence under conditions suitable for hybridization of oligonucleotides whose sequence coincides. substantially with the sequence of the target cDNAs.
  • oligonucleotides that form the population of oligonucleotides comprise a region called “variable” which is what will give rise to the transcripts that will form the siRNAs and a "constant” region, necessary for the subsequent insertion of double-chain oligonucleotides into linearized vectors as well as to incorporate the termination regions of the transcription, so that the transcripts end up in the desired position.
  • variable region can be of any length provided that it allows the subsequent formation of a siRNA and provided that it does not exceed 30 nucleotides, since in that case the response mediated by the protein kinase activated by dsRNA and the RNase L / 2.5 is induced. 'oligoadenylate polymerase resulting in the interruption of all protein synthesis activity and the eventual death of the cell by apoptosis.
  • variable regions of 12 to 30 nucleotides including regions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.
  • variable region it is possible that all positions have an undetermined nucleotide or it is possible to fix some of the positions with a determined nucleotide so that only part of the variable region has a random sequence.
  • the degree of variability of the oligonucleotide population will be determined by the expression 4, where N is the number of indeterminate sequence positions.
  • the variable region consists of 19 nucleotides and all of them are variable.
  • the population of oligonucleotides used in step (a) is usually obtained by chemical synthesis using a single nucleotide precursor at each stage of the synthesis if it is desired to incorporate a fixed nucleotide at the specific position or an equimolar mixture of the precursors of the 4 nucleotides in those stages corresponding to positions in which it is desired to introduce a variable position.
  • the constant regions flanking the variable region can be practically any length, provided that their synthesis is feasible by chemical means but it will be preferable that it be as short as possible since the probability of hybridization by these constant zones is decreased.
  • the constant regions may have from 15 to 18 bp homologous with the ends of the linearized plasmid where it is desired to be cloned by a system based on homologous recombination (Clontech fusion).
  • the olignucleotide constant region has no homology with any open reading pattern in the human genome when applying the Blast-n algorithm.
  • the constant region may contain targets for restriction endonucleases. The target sites allow the generation of cohesive ends in the oligonucleotides after their enzymatic amplification thus allowing their incorporation into a vector with compatible cohesive ends.
  • the restriction targets correspond to enzymes that cut out of their recognition site.
  • enzymes include AIwI, Bbsl, Bbvl, BbvII, BceAl, BciVl, BfuAl, Brnrl, Bpml, BpuEl, Bsal, BseRl, Bsgl, BsmAl, BsmBl, BsmFl, BspMl, EarI, Ecil, Faul, Fokl, Hgal , NboII, MIyI, MnII, Piel, SapI and Sfal.
  • the recognition target for a restriction enzyme consists of the sequence GAAGACN 2 / N 6 , corresponding to the recognition site of the BbvII enzyme.
  • the constant regions of the oligonucleotides that make up the oligonucleotide population contain transcription terminator regions by pol III RNA that allow transcripts generated from each cDNA chain to end at the position desired.
  • the transcription terminator region contains a sequence of 5 adenine residues while the complementary strand contains a sequence of 5 thymine residues.
  • the polyimine or polyadenin regions are preceded (in the 5 'direction in the strand containing the tunnels) by a region rich in GC with palindromic symmetry. Transcription normally proceeds along the polyadenin sequence and ends before reaching the GC-rich region, so that the transcript usually ends at one or more uridine residues.
  • the method of the invention begins by contacting both populations of nucleic acids under conditions suitable for hybridization between the molecules of CDNA and those oligonucleotides whose variable region show a high sequence identity with sequences present in the cDNAs. Suitable conditions are those that allow hybridization of random sequence oligonucleotides with the target cDNA without substantial hybridization of said cDNAs with nucleic acids lacking the sequence complementary to the oligonucleotides of the population.
  • the restrictive conditions can be varied by manipulation of three factors: temperature, salt concentration and formamide concentration. High temperature and low salt concentration increase restrictive conditions. Formamide decreases the melting temperature of DNA, thus decreasing the temperature at which the hybrid can form between two nucleic acid chains.
  • Typical highly restrictive hybridization conditions include the incubation of both nucleic acid populations in 6 X SSC (1 X SSC: 0.15 M NaCl, 0.015 M sodium citrate) and 40% formamide at 42 0 C for 14 hours, followed of one or several wash cycles using 0.5 X SSC, 0.1% SDS at 60 ° C.
  • highly restrictive conditions include those comprising a hybridization at a temperature of about 50 ° -55 ° C in 6XSSC and a final wash at a temperature of 68 ° C in 1-3XSSC.
  • Moderate restrictive conditions include hybridization at a temperature of about 50 ° C to about 65 ° C in 0.2 or 0.3 M NaCl, followed by washing at about 50 ° C to about 55 ° C in 0.2X SSC, 0.1% SDS (dodecyl sodium sulfate).
  • the invention contemplates variations of the conditions described above so that cDNAs can discriminate those oligonucleotides with a high sequence identity from those that do not show such similarity.
  • the cDNA population consists of double stranded polynucleotides, it is necessary to subject said population to a denaturing treatment, preferably in the presence of the oligonucleotide population, in order to allow the separation of the two chains that form the cDNAs.
  • the hybridization reaction between the oligonucleotide population and the cDNA population can be carried out with both molecules being in solution.
  • the cDNA molecule is bound to a solid support, so that after the hybridization reaction, the hybrids can be easily separated from the unhybridized oligonucleotides.
  • the cDNAs have been obtained from a polyT region attached to a solid support, so that when the polyT sequence is extended by reverse transcriptase using the mRNA as a template, the resulting cDNA is coupled to the solid support.
  • the cDNA-oligonucleotide hybrids attached to a support can be easily separated by methods widely known to those skilled in the art, in particular, by centrifugation, filtration or sedimentation.
  • the cDNA-oligonucleotide hybrids are subsequently subjected to a polymerase chain reaction (PCR) using primers capable of specifically recognizing the constant zones of the oligonucleotides.
  • PCR polymerase chain reaction
  • the constant regions of the olignucléotides did not contain targets for restriction enzymes, they can be incorporated during the PCR stage by incorporating said sites into the 5 'region of the primers.
  • PCR amplification of oligonucleotides that have bound to cDNAs is carried out using standard methodology widely known to the person skilled in the art and which is summarized, for example, in Ausubel et al. (Current Protocols in Molecular Biology, Chapter 15, J. Wiley & Sons, Inc. eds., 2003).
  • a population of double-chain oligonucleotides is obtained where the central regions of the oligonucleotides reflect the population of cDNAs derived from a transcriptome associated with a biological process.
  • Said oligonucleotide population must be incorporated into a suitable vector. Cloning can be carried out by known homologous recombination techniques. Alternatively, the oligonucleotide population can be treated with restriction enzymes whose targets appear in the constant region, either because they were included during their initial synthesis or because they were incorporated by the PCR reaction when said target was found in the primers used in the PCR. .
  • the digestion reaction with the specific endonuclease is carried out using the conditions recommended by the manufacturer taking into account the requirements of ionic strength and temperature.
  • the restriction sites present on each side of the variable sequence region are different in order to decrease the frequency of vector relief in the absence of insert.
  • oligonucleotides with protruding and cohesive ends are generated. These oligonucleotides are contacted with a suitable vector that has been previously treated with the same restriction enzyme so that it has cohesive ends compatible with those of the oligonucleotides that make up the population.
  • Suitable vectors in the context of the present invention are all those that include the elements suitable for the expression of the RNA chains that form the siRNA, that is, that contain at least one restriction target that allows the cloning of the oligonucleotides by means of the generation of cohesive ends and that is flanked by two transcription promoters that act in the opposite and convergent direction.
  • a promoter means, in the context of the present invention, a DNA sequence recognized by the synthetic machinery of the cell (endogenous or exogenously supplied) required to initiate the transcription of a gene.
  • any pair of promoters can be incorporated into the cloning vectors in the context of the present invention provided that said promoters are compatible with the cells in which it is desired to express the siRNAs.
  • suitable promoters for the realization of the present invention include, without necessarily being limited, constitutive promoters such as those derived from eukaryotic virus genomes such as polyomavirus, adenovirus, SV40, CMV, avian sarcoma virus, virus hepatitis B, the metallothionein gene promoter, the herpes simplex virus thymidine kinase gene promoter, retrovirus LTR regions, the immunoglobuin gene promoter, the actin gene promoter, the promoter of the EF-lalpha gene as well as inducible promoters in which the expression of the protein depends on the addition of a molecule or an exogenous signal, such as the tetracycline system, the NFkappaB / UV light system, the Cre / Lox system and the heat shock gene promoter, the RNA polymerase II regulatory promoters described in WO / 2006/135436 as well as tissue specific promoters ((for example, the PSA promoter
  • the promoters are RNA polymerase III promoters that act constitutively.
  • RNA polymerase III promoters appear in a limited number of genes such as 5S RNA, tRNA, 7SL RNA and U6 RNAs.
  • type III promoters do not require any intragenic sequence but need sequences in the 5 'direction that comprise a TATA box at positions -34 and -24, a proximal sequence element (proximal sequence element or PSE) between -66 and -47 and, in some cases, a distal element (distal sequence element or DSE) between positions -265 and -149.
  • the type III RNA polymerase III promoters are the Hl and U6 gene promoters of human or murine origin.
  • the promoters are 2 human or murine U6 promoters, a mouse U6 promoter and a human Hl promoter or a human U6 promoter and a mouse Hl promoter.
  • the vectors of the invention contain a reporter or marker gene that allows identifying those cells that have incorporated the vector after having been contacted with it.
  • Reporter genes useful in the context of the present invention include lacZ, luciferase, thymidine kinase, GFP and the like.
  • neomycin resistance gene which confers resistance to aminoglycoside G418, the hygromycin phosphotransferase gene that confers hygromycin resistance
  • the ODC gene which confers resistance to the inhibitor of Ornithine decarboxylase (2- (difluoromethyl) -DL- ornithine (DFMO), the reduced dihydrofolate gene that confers resistance to metrotexate
  • the puromycin-N-acetyl transferase gene which confers puromycin resistance
  • the ble gene that confers resistance to zeocin
  • the adenosine deaminase gene that confers resistance to 9-beta-D-xylofuranosyl adenine
  • the cytosine deaminase gene which allows cells to grow in the presence of iV- (phosphonacetyl) -L- aspartate, thymidine kinase gene, which allows cells to grow in the presence of i
  • the selection gene is incorporated into a plasmid which may additionally include a promoter suitable for the expression of said gene in eukaryotic cells (for example, CMV or SV40 promoters), an optimized translation initiation site (for example a site that follows the so-called Kozak rules or an IRES), a polyadenylation site such as, for example, the polyadenylation site of SV40 or phosphoglycerate kinase, introns such as, for example, the intron of the beta-globulin gene.
  • a promoter suitable for the expression of said gene in eukaryotic cells for example, CMV or SV40 promoters
  • an optimized translation initiation site for example a site that follows the so-called Kozak rules or an IRES
  • a polyadenylation site such as, for example, the polyadenylation site of SV40 or phosphoglycerate kinase
  • introns such as, for example, the intron of the beta-globulin
  • any known vector can be used for the expression of siRNAs according to the present invention.
  • vectors derived from prokaryotic expression vectors such as pUC18, pUC19, Bluescript and their derivatives, mpl8, mpl9, ⁇ BR322, pMB9, CoIEl, pCRl, RP4, phage and shuttle vectors such as pSA3 and pAT28, yeast expression vectors such as 2 micron plasmid type vectors, integration plasmids, YEP vectors, centromeric plasmids and the like, insect cell expression vectors such as pAC series and pVL series vectors , plant expression vectors such as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC series vectors, pMY, pORE and the like and expression vectors in upper eukaryotic cells well
  • oligonucleotides into vectors with compatible cohesive ends is carried out using any of the methods known to those skilled in the art to construct hybrid DNA molecules from two or more fragments, preferably using DNA ligases such as DNA.
  • DNA ligases such as DNA.
  • phage ligase T4 or E.coli under the conditions recommended by the supplier and summarized in sections 3.14, 3.16 and 3.17 in Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley and Sons, eds. 2003. or by homologous recombination.
  • each of the DNA chains is under the transcriptional control of a promoter.
  • under transcriptional control it is understood that the promoter is in the correct location and orientation in relation to the nucleic acid so that it is capable of controlling the initiation of RNA polymerase activity and the expression of the RNA chains that give rise to the siRNA.
  • the invention relates to a method for the preparation of a library of siRNAs specific to a transcriptome associated with a biological process (hereinafter the second method of the invention) comprising:
  • step (b) modify the fragments obtained in step (a) to generate protruding and cohesive ends
  • step (c) inserting the fragments obtained in step (b) into a linearized cloning vector containing cohesive ends compatible with the ends of the oligonucleotides introduced in step (b) wherein said vector It comprises two independent promoters that act in a convergent manner and wherein the oligonucleotide is inserted between both promoters so that one of the promoters controls the transcription of a DNA strand of the fragment and the other promoter controls the transcription of the complementary strand.
  • a population of specific cDNAs of a transcriptome obtained according to any of the methods indicated in relation to the first method of the invention is started.
  • the specific cDNA population of a transcriptome is subjected to a fragmentation treatment.
  • the person skilled in the art will appreciate that the conditions of the fragmentation reaction should be controlled so that the average size of the resulting fragments is adequate for the resulting RNA chains to give rise to a specific siRNA without triggering the apoptotic response mediated by RNase L / 2'5 'oligoadenylate polymerase.
  • the fragmentation reaction must be carried out so that the resulting fragments have an average size of between 12 and 30 nucleotides, preferably about 19 nucleotides.
  • Methods for fragmenting DNA include treatment with endonuclease V, endonuclease Bal 31, nuclease Sl, micrococal endonuclease S.aureus and DNAse I using conditions widely known to the person skilled in the art (see, for example, section 3.12 in Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, eds. 2003).
  • an intermediate stage of fractionation of the obtained fragments may be necessary so that all those outside the appropriate size range for the production of siRNA are eliminated, that is, those fragments of less than 12 nucleotides or more than 30 nucleotides. Preferably, 19 nucleotide oligonucleotides are selected. Fractionation can be carried out by standard techniques such as gel filtration chromatography or preparative electrophoresis.
  • the fragments obtained in the first stage may have blunt ends or protruding ends, depending on the method and conditions employed in the reaction of fragmentation. If the fragment contains protuberant ends, it is necessary to remove them before proceeding to their modification to generate cohesive ends compatible with the cloning vector.
  • the removal of protruding ends is carried out by exonucleases capable of digesting the 5 'and 3' protruding ends of DNA molecules (for example exonuclease VII, exonuclease V and exonuclease RecBCD), exonucleases that act exclusively on protruding ends.
  • exonucleases acting exclusively on 3' protruding ends (e.g. exonuclease I, exonuclease III) using reaction conditions widely known (see section 3.11 in Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, eds. 2003).
  • the cDNA fragments, once treated so that they have blunt ends and have the desired size are modified by ligation to both ends of linkers ("linkers").
  • linker is meant, in the context of the present invention, a double stranded double stranded DNA molecule.
  • Said sequence may include at least one target for a restriction endonuclease so that, after treatment of the adapters or of the products containing said adapters at its end with said restriction enzyme, protruding ends are generated.
  • the linker sequence is not present in any open reading pattern in the human genome or, if it appears, it does so sporadically.
  • the linkers used in the method of the present invention can therefore be provided with at least one target for a restriction endonuclease (or cohesive ends generated by said endonuclease).
  • the linkers also allow the introduction into the sequence of transcriptional terminators that guarantee that the transcripts generated from each of the chains of the DNA fragments have the exact length of said fragments and do not contain additional regions derived from the transcription of the regions from the vector neighboring the insertion site.
  • the restriction sites that can be incorporated into the linkers are chosen based on the same criteria as those used to choose the restriction targets in the constant regions of the oligonucleotides used in the first method of the invention. Therefore, it is interesting to choose restriction targets whose cutting site is outside the recognition site.
  • the restriction targets mentioned above are also suitable for incorporation into the linkers used in the second method of the invention.
  • the linkers include the restriction site for the BbvII endonuclease consisting of the GAAGACN 2 / N 6 sequence.
  • the transcriptional termination sites present in the linkers are preferably the same as those that can be used in the oligonucleotides used in the first method of the invention.
  • Ligation of the fragments of the original cDNA population to the linkers is carried out using methods widely known to the person skilled in the art as set forth above for the first method of the invention.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TMA TMA
  • linkers where no target sites for restriction endonucleases are used, it is necessary to introduce said targets during the amplification stage.
  • oligonucleotide population containing target sites for restriction endonucleases on both sides of the region is available constant, either as a result of the sequence used in the linkers or as a result of the amplification reaction, it is necessary to subject said population of molecules to a restriction endonuclease treatment so as to generate cohesive ends that allow the insertion of oligonucleotides in the vector suitable for the expression of the RNA chains that form the siRNA.
  • Endonuclease treatment is carried out under the conditions provided by the endonuclease provider.
  • the fragments generated from the linkers are separated from the oligonucleotides by techniques based on the different molecular weight of both types of molecules (molecular exclusion chromatography, preparative gel electrophoresis and the like).
  • the oligonucleotide population with cohesive ends is available, it is contacted with a linearized vector containing cohesive ends compatible with the cohesive ends of the oligonucleotide population under the conditions suitable for the ligation reaction.
  • the vectors that can be used for cloning the population of cDNA fragments match those used for the cloning of the double-chain oligonucleotides used in the first method of the invention and have been described in detail above.
  • Both the first and the second method of the invention allow obtaining a population of vectors containing oligonucleotides that can be transcribed to give rise to the two chains of a siRNA.
  • the invention provides a library obtainable by the first or second method of the invention.
  • the library of the invention consists of a population of vectors that, when introduced into a host organism or cell, give rise to the formation of siRNA.
  • the siRNAs resulting from the expression of said libraries must cause the inhibition or silencing of at least one gene that is involved in said process.
  • the invention relates to a method for obtaining vectors for the expression of a siRNA capable of interfering in a biological process comprising the steps of
  • step (b) introducing into a cell or organism a library of the invention prepared from a cDNA population derived from a transcriptome associated with said biological process (b) identifying those cells or organisms in which an indicative phenotypic alteration manifests of an alteration in said biological process and (c) rescue the vector or population of vectors from the library from the cells or organisms identified in step (b).
  • the introduction of the vectors that form the library according to the present invention in the cell where it is desired to identify the alteration in a certain biological process can be carried out using any method known to the person skilled in the art. Suitable methods include transfer mediated by a vector (for example, by infection with a recombinant virus or by transfection with a viral vector), methods in which DNA complexes are used to be introduced into the cells with proteins, lipids or other types of polymers (lipofection, DEAE-dextran, polibreon) and methods that allow the introduction of naked DNA into the cell (electroporation, using biolistic methods, microinjection).
  • the invention contemplates the use of any type of cell into which an expression vector can be introduced.
  • embryonic cells oocytes, sperm cells, embryonic stem cells
  • adult cells undifferentiated cells (fetal cells, tumor cells), differentiated cells, dividing cells, senescent cells, cultured cells and the like.
  • the polynucleotides introduced into the cell can be maintained transiently or stably in the host cell.
  • the gene that allows the selection of cells can be provided as part of the same vector that contains the construction object of the invention or, alternatively, can be provided separately by co-transfection with a second plasmid containing said resistance gene.
  • the plasmid containing the DNA construct is contributed to the transfection mixture in a molar excess with respect to the resistance gene so that for each event of integration of the resistance gene there is a high probability of integration of the gene. which contains the promoter under study.
  • the plasmid containing the DNA construct is provided in an excess of at least 5 times with respect to the vector containing the resistance reporter.
  • the siRNA library is in a lentiviral vector using a controlled infection multiplicity of 1 or less, so that a population of cells in which about 100% thereof has been obtained is obtained. infected. This achieves a very high frequency of incorporation.
  • Resistance markers suitable for selecting cell lines that have integrated the construction into the genome include positive selection markers such as the geneticin resistance gene, the neomycin resistance gene, which confers resistance to the G418 aminoglycoside, the gene of the hygromycin phosphotransferase that confers hygromycin resistance, the ODC gene, that confers resistance to the ornithine decarboxylase (2- (difluoromethyl) -DL-ornithine (DFMO) inhibitor, the dihydrofolate reductase gene that confers resistance to metrotexate, the gene from puromycin-N-acetyl transferase, which confers resistance to puromycin, the ble gene that confers resistance to zeocin, the adenosine deaminase gene that confers resistance to 9-beta-D-xylofuranosyl adenine, the cytosine deaminase gene, which allows cells to grow in the presence of iV- (phospho
  • the selection gene is incorporated into a plasmid which may additionally include a promoter suitable for the expression of said gene in eukaryotic cells (for example, CMV or SV40 promoters), an optimized translation initiation site (for example a site that follows the so-called Kozak rules or an IRES), a polyadenylation site such as, for example, the polyadenylation site of SV40 or phosphoglycerate kinase, introns such as, for example, the intron of the beta-globulin gene.
  • a promoter suitable for the expression of said gene in eukaryotic cells for example, CMV or SV40 promoters
  • an optimized translation initiation site for example a site that follows the so-called Kozak rules or an IRES
  • a polyadenylation site such as, for example, the polyadenylation site of SV40 or phosphoglycerate kinase
  • introns such as, for example, the intron of the beta-globulin
  • the process of selecting cells that contain the DNA construct of interest stably integrated into the genome is carried out by a conventional selection process (see for example Ausubel, FM et al., Current Protocols in Molecular Biology (1997) 9.5.1-9.5.19).
  • the cells are transfected with the vector and, after a recovery period, they are allowed to grow in a selective medium (either a medium containing the antibiotic against which the reporter confers resistance or a minimum medium containing the antimetabolite against to which the reporter confers resistance).
  • a selective medium either a medium containing the antibiotic against which the reporter confers resistance or a minimum medium containing the antimetabolite against to which the reporter confers resistance.
  • recombinases can be used, in particular the Cre / Lox system.
  • Organisms that can be used in the context of the present invention include animals (vertebrates and invertebrates), plants (monocots and dicots), protists (algae, ciliates, diatoms) and fungi (including multicellular and unicellular fungi).
  • animals and, even more preferably, non-human mammals are employed.
  • Methods for the generation of mammals, fish, birds and transgenic insects are employed.
  • Such methods include the use of retroviral particles, in particular lentivirals, which carry the constructs from the library of the invention for the infection of cells preferably oocytes, zygotes or early embryos, the transfection of the vectors of the library of the invention into cells Trunks of embryonic origin and then incorporate said cells into an embryo or the generation of oocytes or sperm cells by in vitro differentiation of embryonic stem cells that contain the library vectors integrated into their genome.
  • retroviral particles in particular lentivirals, which carry the constructs from the library of the invention for the infection of cells preferably oocytes, zygotes or early embryos
  • Alternative methods include the exchange of recombinase-mediated constructs for which the construct to be integrated is flanked by non-identical recombinase target sites, such as loxP or Iox2272 that can be recognized by a recombinase, such as, for example, Cre de so that the siRNA expression cassette is introduced by homologous recombination in a region of the chromosome also flanked by loxP or Iox2272 sites.
  • non-identical recombinase target sites such as loxP or Iox2272 that can be recognized by a recombinase, such as, for example, Cre de so that the siRNA expression cassette is introduced by homologous recombination in a region of the chromosome also flanked by loxP or Iox2272 sites.
  • Transgenic non-human animals carrying the vectors of the invention are obtained by introducing a nucleic acid molecule or a vector from the library of the invention into the nucleus of a cell, preferably an embryonic cell, replacing the nucleus of a oocyte, zygote or an early embryo with said nucleus and transferring said oocyte, zygote or early embryo to a mother or cultivating said oocyte, zygote or early embryo and transferring said embryo to a mother to leave the embryo that reaches term.
  • Embryonic stem cells are preferably used to obtain non-human transgenic organisms.
  • the AB-I murine origin embryonic cell line grown on layers of mitotically inactive feeder cells SNL7617 can be used.
  • Other trunk embryonic cell lines include lines E14, D3, CCE and AK-7.
  • the phenotypic alteration to be detected would be an increase in resistance to said potentially carcinogenic agents; if the process to be studied is cell proliferation, the phenotypic alterations indicative of an alteration in said process would be, among others, an increase or decrease in the duplication rate, the ability of the cells to overcome a mitotic blockage induced by agents exogenous chemicals (for example, nocodazole); If the biological process to be studied is the sensitivity of a cell or organism to a pathogen, the phenotypic alteration to be detected would be an increase or decrease in the sensitivity of the cell or organism to infection by the pathogen.
  • phenotypic alterations that can be used for the detection of genes involved in a given biological process is varied and essentially depends on the type of biological process that you want to study.
  • morphological, biochemical changes enzyme activity, alteration in the levels of certain metabolites, protein patterns by electrophoresis in two-dimensional gels, pattern of phosphorylated proteins
  • behavior chemotropism, phototropism in the case of unicellular organisms or changes can be detected in spatial memory, aggressiveness and the like in the case of multicellular organisms.
  • the biological process to be studied is cell proliferation and the phenotypic alteration that is detected is the ability of cells to overcome the mitotic blockade in nocodazole-induced metaphase at a concentration of at least 1 ⁇ M.
  • siRNA gene can be rescued by PCR from the DNA of the identified cells or organisms or by rescue of the viral genome in case the vector used to express the siRNA was a viral vector.
  • the digestion of the PCR products obtained from the cells that show a phenotypic alteration allows their cloning into previously linearized vectors and that have cohesive ends compatible with the ends generated in the PCR products. In this way a unique vector is obtained that is capable of generating siRNA capable of specifically inhibiting a biological process.
  • the process described above may result in the isolation of a single clone or the isolation of a subpopulation of clones from the original population. If this is the case, the process can be repeated successively using the subpopulation of clones obtained after the first isolation process as the starting material. After one or more additional purification cycles, a single clone or a reduced population of clones will be obtained that will encode a specific siRNA for the original biological process.
  • the clone thus obtained can be used to inhibit said biological process in vitro or in vivo.
  • the siRNA can be synthesized in vitro from the clone that encodes it by transcription in the presence of the RNA polymerase promoters to thus give rise to the two RNA strands that will hybridize to give rise to the active siRNA.
  • the siRNA can be generated in vivo by expression in a heterologous organism of the clone that encodes the siRNA from where they can be subsequently extracted for purification.
  • the siRNAs can be generated in bacteria, as described in WO / 2006/130976.
  • the siRNA of interest can be generated in vivo by transfection in a cell or by incorporation into the genome of a Transgenic organism of the clone that contains the elements necessary for the expression of the two RNA chains that form the siRNA.
  • the libraries of the invention allow the identification of genes involved in a certain biological process. Since these libraries are obtained from a population of enriched cDNAs for a given biological process, the degree of diversity of the library is lower than the theoretical number of 4 19 that would be possible if the
  • the invention relates to a method for the identification of a gene involved in a biological process comprising: a) introducing into a cell a library of the invention, b) identifying those cells in which alterations are observed phenotypic indications of alterations in said biological process, c) rescue from the cell identified in stage (b) the vector or vectors from the library introduced in step (a) d) sequence the central region of the vector insert or isolated vectors from the library and e) identify genes that show a degree of sequence similarity with said sequence of the central region of the vector where genes that have a high sequence identity with the central region of one or more inserts are candidates to be involved in the biological process
  • the steps (a) to (c) coincide with the steps of the method of obtaining a vector described above.
  • the region of the insert that is located between the two promoters of the vector is sequenced. Sequencing can be carried out using methods widely known to those skilled in the art such as the Sanger method using dideoxynucleotides, sequencing by hybridization (sequencing-by-hybridization or SBH), nanopore sequencing, sequencing by synthesis (sequencing-by- syntehsis or SBS), pyrosequencing and the like. Primers suitable for the sequencing reaction can be obtained from sequences flanking the insert encoding the siRNAs, either using promoter sequences or using sequences around the insertion site.
  • the sequence of the siRNAs that are capable of causing a phenotypic alteration in a cellular system or in an organism is known, it is possible to analyze sequence databases to identify the nucleic acid in the cell or organism that is inhibited by said siRNA and, therefore, potentially involved in the biological process under study.
  • the identification of genes and open reading patterns in the genome of a cell with sequence identity with the siRNA is carried out using algorithms known to the person skilled in the art, such as the BLAST algorithm (Altschul et al., Nuc. Acids Res., 25: 3389-3402 (1977)), BLAST 2.0 (Altschul et al., J. Mol. Biol, 215: 403-410 (1990) or FASTA (WR Pearson and DJ Lipman (1988), Proc.Natl.Acad.Sci. USA 85: 2444-2448).
  • the siRNA sequence can be used to construct probes or primers that allow the isolation of the target mRNAs.
  • the siRNA sequence can be used for the preparation of radioactively labeled probes for screening cDNA and genomic libraries.
  • the siRNA sequence can be used to synthesize primers that can be used to amplify cDNAs from a cDNA or genomic library by techniques such as 5'-RACE or 3'-RACE.
  • the biological process that is under study by the gene identification method of the present invention is cell proliferation. This process would consist of distinguishing those that avoid and / or survive a long metaphase stop (24-30 hours) caused by high concentration nocodazole (l ⁇ M). Cells stopped in Metaphase for a long period end up inducing cell death by apoptosis (Weaver, BA & Cleveland, DW, 2005, Cancer CeIl 8, 7-12). Those cells that by loss of expression of a gene continue with the cell cycle even in the presence of nocodazole and therefore survive or do so by means of a resistance to induce apoptosis will be selected. From In this way, the scrutiny experiment will consist of treating the cells for 24 or 30 hours with nocodazole.
  • the invention in another aspect, relates to a method for altering a specific biological process in a cell or organism that comprises introducing into said cell or organism at least one vector of the library of the invention, wherein said library has been obtained from of a cDNA population derived from a transcriptome associated with said biological process.
  • the biological process under study is cell proliferation.
  • kits comprising, in one or more containers, the reagents necessary to carry out the methods according to the present invention.
  • the invention relates to a kit (hereinafter the first kit of the invention) for the preparation of a library of the invention by the first method of the invention comprising
  • each oligonucleotide comprises a central region of random sequence flanked by regions of constant sequence
  • a cloning vector comprising one or more restriction targets that are flanked by two independent promoters that act in a convergent manner.
  • the constant regions of the oligonucleotides that form the defined population (a) in the first kit of the invention contain a target for a restriction endonuclease that may be the same or different in both constant regions and that is or are identical or compatible with the restriction targets found in the vector defined in (b).
  • the first kit of the invention additionally contains a pair of primers that allow amplification of the oligonucleotides that form component (a) of the kit of the invention through its constant regions and which comprise targets of restriction identical or compatible with those found in the vector defined in (b).
  • the first kit of the invention comprises contacting a specific cDNA population of a given transcriptome with the population of oligonucleotides that form component (a) under conditions suitable for hybridization between the variable region of the oligonucleotides and those cDNAs showing substantial sequence homology with said oligonucleotides, followed by elution of the selected oligonucleotides and incorporating the vector defined in component (b).
  • oligonucleotides Since the amount of oligonucleotides that are obtained after affinity selection over the cDNA population is very small, it is convenient to amplify the oligonucleotides using a pair of primers that show sequence identity with the constant regions of the oligonucleotide forms and that contain identical or compatible restriction targets with those found in the vector defined in (b).
  • the amplification products and the vector are treated with the same restriction endonuclease or with restriction endonucleases that generate compatible ends, thus allowing the cloning of the oligonucleotides into the vectors.
  • the invention relates to a kit (hereinafter the second kit of the invention) for the preparation of a library of the invention comprising (a) an endonuclease
  • a cloning vector comprising one or more restriction targets that are flanked by two independent promoters that act convergently.
  • the linkers that are part of the second kit of the invention contain a target sequence for a restriction endonuclease that is identical or compatible with at least one of the restriction targets found in the vector defined in ( C).
  • the second kit of the invention additionally comprises a pair of primers whose sequence is complementary to the linker sequences of component (b) and which allow amplification of molecules resulting from ligation of linkers on both sides of a DNA chain.
  • the second kit of the invention involves contacting a specific cDNA population of a transcriptome with the endonuclease (component (a)) that generates fragments of the cDNAs.
  • Such fragments are contacted with the linkers so that a linker is added to both sides of each fragment.
  • the linkers contain restriction targets, the resulting molecules can be treated with said restriction target so as to incorporate them into the compatible vector.
  • Said primers incorporate in the region 5 'restriction targets that allow the insertion of the amplified fragments into the vector of choice after treatment of both with identical restriction endonucleases or that generate compatible ends.
  • the vectors included in the kits of the invention include constitutive promoters of RNA polymerase III.
  • the constitutive promoters of RNA polymerase III are HI or U6 promoters of human or murine origin.
  • the first or second kit of the invention additionally comprises the reagents necessary to obtain a population of specific cDNAs of a transcriptome.
  • Kit means, in the context of the present invention, a product containing the various reagents necessary to carry out the first and second methods of the invention packaged to allow transport and storage.
  • Suitable materials for packaging kit components include glass, plastic (polyethylene, polypropylene, polycarbonate and the like), bottles, vials, paper, envelopes and the like.
  • the kits of the invention may contain instructions for simultaneous, sequential or separate administration of the various components found in the kit. Said instructions may be in the form of printed material or in the form of an electronic support capable of storing instructions so that they can be read by a subject, such as electronic storage media (magnetic discs, tapes and the like), optical media (CD- ROM, DVD) and the like. Additionally or alternatively, the media may contain Internet addresses that provide such instructions.
  • a 73 base oligonucleotide was designed consisting of a constant sequence of 27 nucleotides at each end and a central random sequence of 19 nucleotides.
  • the end sequences contain the GAAGACN 2 ZN 6 restriction site recognized by the BbvII enzyme and have no homology (17 nucleotides in a row) with no open reading pattern in the human genome, when applying the Blast-n algorithm. Digestion with BbvII results in prominent 5 'ends that would match those of the vector to be used.
  • the lentiviral vector pFIV-Hl / U6-EGFP [System biosciences] based on the pFIV-copGFP vector has been chosen for the construction of the library, since lentiviral vectors are the most effective for infecting mammalian cells, including quiescent cells , primary, mother and differentiated.
  • This vector comes from the feline virus of the Immunodeficiency (IVF) is biologically safer than third generation HIV vectors.
  • the Hl and U6 promoters of the vector oriented in opposite directions flank the designed oligonucleotide and have the termination signal (T 5 ) "upstream" of the early transcription ( Figure 2).
  • T 5 termination signal
  • Figure 2 The resulting double-chain siRNA product does not require intracellular processing. Since the molecule does not contain the "hairpin" structure, it is more stable during the propagation stage in Escherichia coli, and ensures greater complexity of the resulting library.
  • constructs have been transfected into HEK 293T cells [ACTT- American Type Culture Collection-] together with plasmids pFIV-34N [System biosciences], which contains the structural, regulatory and replicative genes necessary for lentivirus production, and p VS VG [ System biosciences], which expresses the envelope glycoprotein of the vesicular stomatitis virus, and the resulting virus can infect any eukaryotic cell.
  • a selection of molecules corresponding to specific mRNA sequences present in the nocodazole exposed cell has been carried out.
  • mRNA was isolated. Similarly, mRNA was isolated from HCTl 16 cells without treatment. This mRNA remained immobilized on the oligodT magnetic balls and was re-transcribed to cDNA bound to said balls. The mRNA isolated from the treatment with nocodazole was hybridized to the cDNA obtained from the cells not exposed to the drug and in this way all the transcript that had not been specifically induced in the presence of nocodazole was bound to the cDNA, the mRNAs being isolated by elution do not hybridize (75% and 60% subtraction, in two experiments performed). This subtracted mRNA it was re-isolated by binding to the oligodT magnetic balls and served as a template for the corresponding cDNA chain by retrotranscription.
  • the mRNA was removed and the cDNA population was hybridized in a 0.5% solution of SDS and 6x SSC, at 38 0 C or at 50 0 C with 5 (110 ⁇ g) and 20 (440 ⁇ g) nmoles of random oligonucleotides, respectively.
  • 464 ng of the 110 ⁇ g (0.4%) and 120 ng of the 440 ⁇ g (0.027%) used in the hybridizations remained paired with the cDNA molecules.
  • the oligonucleotides were eluted and amplified by primers complementary to the constant ends of the oligonucleotides ( Figure 4).
  • the amplification conditions were as follows:
  • Two hybridization temperatures were used to obtain two groups of oligonucleotides, some less specific but encompassing a greater number of different siRNAs for each cDNA and others more specific but with a lower representation of different siRNAs for each cDNA.
  • the amplification products once digested with the BbvII enzyme ( Figure 1), were passed in a buffer through a Streptavidin resin (Dynabeads® M-270 Streptavidin, Dynal Biotech, Oslo, Norway) to which the fragments bind of the ends of the cut, since the primers used are biotinylated.
  • the 19 bp eluted DNA was cleaned through a Sephadex G-25 column [Sigma] and ligated to the vector pFIV-Hl / U6-EGFP [System Biosciences].
  • the resulting constructs were transformed by electroporation into competent bacteria DH5 ⁇ [ACTT-American Type Culture Collection-] (yield of 2-3 10 9 / ⁇ g of plasmid). From the recombinant colonies, the corresponding plasmids were massively obtained which, together with the aforementioned plasmids pFIV-34N and pVSV-G, were transfected into HEK293T [ACTT-American Type Culture Collection-] cells to obtain lentiviral particles .
  • the in vitro functional test for cell selection consists in distinguishing those that bypass and / or survive a long stop in Metaphase (24-30 hours) caused by high concentration nocodazole (l ⁇ M). Cells stopped in metaphase over a long period end up inducing cell death by apoptosis (Weaver, B. A. & Cleveland, D. W., 2005, CeIl Cancer 8, 7-12). Those cells that by loss of expression of a gene continue with the cell cycle even in the presence of nocodazole and therefore survive or do so by means of a resistance to induce apoptosis are selected. Thus, the scrutiny experiment consists of treating the cells for 24 or 30 hours with nocodazole.
  • HCTl 16 cells [ACTT- American Type Culture Collection-] exposed to nocodazole l ⁇ M for 24 hours, they were only able to form 10-20 clones while the HCTl 16 Mad2 +/- line was able of forming 250 clones.
  • These clones have a phenotype similar to Mad2 +/- either by deregulating the Metaphase checkpoint or by being able to delay or cancel intracellular apoptosis signaling.
  • the siRNAs that cause the cell proliferation inducing phenotype are amplified with primers of the U6 and Hl promoters and checked against the databases to know which gene has been attenuated in their expression.
  • a good selection system will be needed to identify the positive clones of the negative ones as well as a good definition of the subtraction conditions where you will have to there is an induced mRNA and a basal state mRNA.
  • the subtraction protocol will isolate the specific mRNAs from a condition and target of the siRNAs that will be part of the libraries.

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Abstract

La présente invention concerne des procédés permettant d'obtenir des génothèques d'ARNsi à partir d'une population de polynucléotides correspondant à un transcriptome associé à un procédé biologique déterminé dans lequel les chaînes d'ARN formant l'ARNsi proviennent de la transcription de la chaîne sens et antisens d'un ADN double chaîne par l'action de deux promoteurs convergents. Cette invention concerne des trousses permettant de mettre en oeuvre ces procédés, ainsi que des procédés permettant d'identifier des gènes utiles pour un procédé biologique déterminé et qui consistent à utiliser les génothèques susmentionnées.
PCT/ES2008/000758 2007-12-07 2008-12-04 PROCÉDÉS ET TROUSSES POUR LA PRÉPARATION DE GÉNOTHÈQUES D'ARNsi SPÉCIFIQUES D'UN TRANSCRIPTOME PAR TRANSCRIPTION CONVERGENTE WO2009071722A1 (fr)

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