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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
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/90—Stable introduction of foreign DNA into chromosome
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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
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/40—Fish
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/02—Animal zootechnically ameliorated
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Definitions

• the invention relates to an in vitro method for introducing into an oocyte or egg a targeted genomic modification and a method for performing random insertion into the genome of a host cell.
• Transgenesis is a technique of molecular genetics by which exogenous DNA is introduced into the genome of a multicellular organism and is transmitted to the offspring of the latter. This transmission to the offspring imposes the stable integration of said DNA into the genome of the embryo and this at an early stage of development.
• transgenesis techniques are the microinjection of naked DNA into a mammalian egg, which in a number of cases leads to the integration of part of the microinjected DNA molecules in the genome of the egg.
• Other techniques can be used for transgenesis, and in particular techniques for introduction of exogenous DNA into a living cell which are well known to those skilled in the art, in particular electroporation, transfection with precipitates of calcium phosphate, liposomes or modified lipids such as lipofectamine® (IN VITROGEN).
• the exogenous DNA In the case of targeted integration of exogenous DNA into the genome, it is necessary to use the homologous recombination mechanism.
• the exogenous DNA In this case, the exogenous DNA must have homologous nucleic acid sequences with those present at the targeted integration site in the genome.
• these homologous recombination mechanisms operate at an extremely low frequency in most organisms.
• the inventors have demonstrated that it was possible, by introducing an exogenous nucleic acid sequence and meganuclease I-SceI into an egg, which has in its genome an I-SceI site flanked by homologous sequences. to the exogenous nucleic acid sequence, to obtain an egg with a targeted genomic modification corresponding to the homologous recombination insertion of an exogenous nucleic acid sequence at the genomic I - Scel site.
• the discovery of the inventors makes it possible to demonstrate that, if the homologous recombination mechanism that uses a meganuclease can be used in vivo, the said mechanism can also be carried out directly in oocytes or eggs with sufficient efficacy, and this without compromising the achievement the development program of that body.
• the method of the inventors then makes it possible to obtain an egg or an oocyte having a targeted genomic modification, and potentially to obtain directly a mature genetically modified organism with such a targeted genomic modification, and this in all of its cells.
• Said targeted genomic modification may then correspond to a deletion or an insertion, in particular the insertion of a mutated sequence with respect to the wild-type sequence.
• the cell of the egg or oocyte contains a large cytoplasm compared to that of a normal cell which makes it difficult to access the nucleus that contains the genetic material.
• a membrane vitelline membrane
• a chorion present specifically around the eggs to protect them limits access to the cell.
• the method according to the invention makes it possible to directly obtain transgenic animals, all of whose cells exhibit the targeted genomic modification.
• the prior art suggested to those skilled in the art the isolation of such cells and in no case the process according to the invention. which makes it possible to obtain transgenic animals directly from the egg for such organisms.
• the plasmid was injected in very large amounts and simultaneously with meganuclease I-SceI, which is unstable when it is not fixed on its site.
• This large amount of I-Sce I sites and this co-injection, which facilitated the stabilization of the meganuclease, did not in any way allow to predict the homologous recombination frequency obtained in the presence of a rare site, because localized at most to a few copies in genomic DNA, and moreover difficult to access because of the compact structure of genomic DNA.
• the chromatin structure and the scarcity of the sites could legitimately be expected not to allow meganuclease access to one of its sites until it has been degraded.
• a first object of the invention is an in vitro method of producing nonhuman vertebrate eggs or oocytes having a targeted genomic modification comprising:
• egg we mean a single cell resulting from the fertilization of a female gamete by a male gamete and which contains all the potentialities necessary for the formation of a new organism. More simply, the egg corresponds to an embryo at the one - cell stage.
• oocyte a female germ cell obtained during the oogenesis phase of maturation.
• the method according to the invention is an in vitro method for producing nonhuman vertebrate eggs having a targeted genomic modification.
• nonhuman vertebrates that can be used in eggs or oocytes in the process according to the invention include mammals such as rodents, sheep, cattle or nonhuman primates, reptiles, amphibians like Xenopus, birds like hen, insects like fly and fish like zebrafish or Medaka.
• the egg or oocyte used in the method of the invention is an egg or a fish oocyte, such as salmon, trout, tuna, halibut, catfish, zebrafish, medaka, carp, stickleback, astyanax, tilapia, goldfish, bass, sturgeon or loach.
• the egg or the oocyte used is an egg or a oocyte of zebrafish (Danio rerio) or Medaka (Orizias latipes).
• the method according to the invention also applies without difficulty to other aquatic species such as frog, xenopus, shrimp, sea urchin.
• recognition site is meant a specific nucleic acid sequence which has a length of at least 12 base pairs to which said endonuclease specifically binds and which, after the attachment of the endonuclease to the latter, induction of a double - strand break in the DNA by said endonuclease.
• said recognition site corresponds to a specific nucleic acid sequence of at least 16 base pairs, and particularly preferably at least 18 base pairs.
• nucleic acid sequence is meant a DNA sequence, preferably a double-stranded DNA sequence.
• the identification step can be performed using techniques well known to those skilled in the art. This identification step can use, by way of example, Southern or PCR techniques, on the genomic DNA isolated from the egg or the oocyte obtained, using a probe or specific primers respectively.
• said identification step may use techniques for detecting the activity of said reporter gene. Such detection techniques are dependent on the reporter gene used and are well known to those skilled in the art.
• said identifying step corresponds to a step of culturing said oocyte in a suitable medium. The culture conditions for such a step are a function of the selection gene used and are well known to those skilled in the art.
• said specific nucleic acid sequence for said endonuclease corresponds to the binding consensus sequence determined for said endonuclease or to a sequence derived from said consensus sequence.
• certain endonucleases are able to bind to sequences that do not have a perfect identity with their consensus sequence and to carry out, following this fixation, a double-strand break at the latter or in its neighboring regions.
• said specific sequence has more than 90% identity with said consensus sequence, preferably more than 95%, and particularly preferably more than 98% with said consensus sequence.
• Percentage of identity means the percentage of nucleic acids of identical nature and position between said specific sequence and said determined binding consensus sequence for said endonuclease.
• the endonuclease used it is possible to obtain a double - strand break in the DNA, either at said recognition site specifically, or in the neighboring sequences of said recognition site, preferably at less than 100 base pairs of said site. of recognition, preferably less than 50 base pairs, and particularly preferably less than 20bp.
• the double-strand break induced by the endonuclease is localized at its recognition site in the genomic DNA.
• Said recognition site may be present in the genomic DNA of wild individuals or it may have been introduced into said genomic DNA by transgenesis.
• said recognition site has been introduced by transgenesis into the genomic DNA of said egg or oocyte.
• the introduction of this recognition site into the genomic DNA could be carried out in a targeted or random manner.
• said introduction by transgenesis could be carried out either in said oocyte or egg used in the process according to the invention, or in an oocyte, an egg or a cell from which or from which a sexually mature organism was able to develop and from which is derived 1 Oocyte or the egg used in the process according to the invention.
• the introduction of said recognition site is carried out in a targeted manner.
• targeted introduction can be carried out by homologous recombination according to techniques well known to those skilled in the art.
• COHEN-TANNOUDJI et al. (1998, supra) describes the injection into the nucleus of a vector which contains a selection gene and a recognition site for a meganuclease, which are flanked by nucleic acid sequences homologous to the target sequences of the genomic DNA. .
• the cells having integrated the construction at the desired position in the genomic DNA are identified, in particular by Southern blotting or by PCR. Recombination rates being low in these conditions, the number of cells with targeted insertion is extremely small.
• the targeted introduction of the recognition site for said endonuclease into the genomic DNA is carried out by homologous recombination.
• the introduction of said recognition site is performed randomly.
• different techniques can be used.
• CHOULIKA et al (1998, Mol.Col Biol., Vol.15 (4), p: 1968-1973, 1995) discloses the use of a retroviral vector for the integration of recognition for meganuclease I-Sce I in genomic DNA.
• PCT application WO 03/025183 describes another method of random integration of recognition sites for meganuclease I-Sce I in the genomic DNA of a fish egg by micro-injecting simultaneously in its nucleus meganuclease I-Sce I and a DNA fragment which has a reporter gene flanked by two recognition sites for I-Sce I. It is also possible to use the method of random integration of nucleic acid sequences according to the invention described in the examples. .
• the random introduction of the recognition site for said endonuclease into the genomic DNA is carried out by the random integration method of nucleic acid sequences described in the patent application WO 03/025183 or by the integration method. random sequence of nucleic acid sequences described in the examples.
• Endonucleases capable of binding to a specific sequence of at least 12 base pairs, inducing a consecutive double - strand break of the DNA at said specific sequence, or in its surrounding regions, and ultimately driving repairing said double-strand breakage by a mechanism homologous recombination are known to those skilled in the art. Examples of such endonucleases include meganucleases.
• Meganucleases are a family of enzymes that perform double strand DNA cleavage at a very low frequency. Indeed, said meganucleases have recognition sites of 12 to 40 base pairs, whereas the conventional restriction enzymes have recognition sites generally of the order of 4 to 8 base pairs. The probability of presence of such a recognition site in the genomic DNA is therefore extremely low. Meganucleases are also well characterized from a structural and mechanistic point of view. Meganucleases fall into four distinct families based on conserved amino acid patterns.
• the dodecapeptide family (dodecamer, DOD, DOD, D1-D2, LAGLI6DADG, P1-P2) is the most important family with more than 150 sequences grouped according to whether they have one (I-Ceul, I-Crel) or two copies (I-ChuI, I-Csml, I-Dmol, I-PanI, I-SceI, I-Scell, I-SceIII, I-SceIV, F-SceI, F-Scell, PI-Aael, PI-Apel, Pl -Col, Pl-CirI, Pl-CtrI, PI-Dral, PI-MavI, PI-MfII, PI-Mgol, PI-Mjal, PI-MkaI, PI-MIeI, PI-MtUI, PI-MtUHI, Pl-PablII , PI-Pful, PI-PhO1, PI-Pk
• the meganucleases with a dodecapeptide have a molecular weight of the order of 20 kDa and act as a homodimer.
• the meganucleases with two dodecapeptides have a molecular weight of 25 to 50 kDa, with 70 to 150 residues between the two units, and are active as monomers.
• the GIG family has a complete conserved motif KSGIY-X 10/11 -YIGS (I-NcrI, I-NcrII, I-PanII, I-TevI) or partial (I-TevII) and the enzymes of this family cut DNA in a site different from their site of recognition.
• the HC family has sequences rich in histidines and cysteines (I-Pop1, I-DirI, I-Emul, I-HmuII) with generally a conserved sequence that corresponds approximately to "SHLC-G-G-H-C".
• the best characterized meganuclease for this family is the enzyme I-Ppol.
• the HNH family has a consensus sequence "HH-N-H-H” in a window of 35 residues (I-TevIII) and particular DNA cleavage properties.
• the endonuclease used is a meganuclease or an enzyme derived from such a meganuclease, which can be synthetic.
• I-Ceul meganucleases I-CreI, I-ChuI, I-Csml, I-Dmol, I-PanI, I-SceI, I-Scell, I-SceIII can thus be mentioned, 1-SceIV, F-Sce I, F- Scell, PI-Aael, PI-Apel, PI-CeuI, PI-PI-ciri f r CTRI DraI PI, PI-MAVI, PI-MFII, PI-Mgol, PI- Mjal, PI-MkaI, PI-MIeI, PI-MtUl, PI-MtUHI, Pl-PablII, PI-Pfui, PI-PhO1, PI-PkO1, PI-PspI, PI-Rmal, Pl-SceI, PI-SspI, PI-TfuI, PI-
• meganuclease is meganuclease I-SceI described in US Pat. No. 6,238,924.
• meganuclease derivative or enzyme derived from a meganuclease is meant a recombinant protein having sequences of a wild meganuclease and which is capable of recognizing a recognition site different from said wild-type meganuclease and / or to perform a double-strand break of the DNA at a different position or by a different mechanism from said wild-type meganuclease.
• Said meganuclease derivative also makes it possible to cause the repair of said double - strand break by a homologous recombination mechanism.
• meganucleases derived include, in particular, recombinant meganucleases whose DNA binding domain is derived from other DNA binding proteins such as IIS-type restriction endonucleases or transcription factors.
• recombinant meganucleases derived from there may also be mentioned recombinant meganucleases which have one or more nuclear localization sites absent wild meganucleases from which they are derived.
• the endonuclease may be introduced exogenously into the egg or oocyte in various forms, namely in the form of a protein or in the form of a nucleic acid sequence allowing the expression of said endonuclease in said egg or oocyte.
• the endonuclease is introduced into the egg or the oocyte in the form of a protein.
• Techniques for introducing such an endonuclease in the form of a protein are known to those skilled in the art. By way of example of such techniques, mention may be made in particular of microinjection.
• the concentration of said endonuclease introduced exogenously is between 0.1 and 5 units per ⁇ l and per egg or oocyte, preferably between 0.5 and 2.5 units per ⁇ l, and particularly preferably between 1 and 2 units per ⁇ l.
• volume injected per egg or oocyte is part of the general knowledge of those skilled in the art and is of the order of 10% of the volume thereof.
• the volume likely to be injected into a zebra or medaka egg or oocyte is between 300 ⁇ l and 1 ⁇ l.
• the amount of nucleic acid introduced per egg or oocyte is then between 0.3 ⁇ 10 -4 and 5 ⁇ 10 -3 units, preferably between 1.5 ⁇ 10 -4 and 2.5 ⁇ 10 -3 units, and particularly preferably between 0.3 ⁇ 10 -2. "3 and 2x10 " 3 units.
• the endonuclease is introduced in the form of a molecule of nucleic acids allowing the expression of said endonuclease in said egg or oocyte.
• the nucleic acid molecule then comprises an open reading phase, encoding the endonuclease, under the control of regulatory sequences for the expression of said endonuclease in the egg or oocyte.
• nucleic acid molecule By nucleic acid molecule is meant both DNA, RNA and hybrid DNA / RNA molecules, which may be in single or double stranded form.
• nucleic acids As an example of a molecule of nucleic acids that can be used in the process according to the invention, mention may be made of an endonuclease-encoding mRNA molecule or an expression vector comprising an open reading phase coding for said endonuclease.
• expression vector is meant a molecule of nucleic acids capable of transporting and allowing the expression of a nucleic acid sequence of interest to which it is operably linked.
• Such an expression vector contains a promoter sequence allowing expression of said endonuclease in the egg or oocyte.
• promoters By way of example of such promoters, mention may be made in particular of the ⁇ -tubulin or ⁇ -actin promoter, or else the strong constitutive promoters well known to those skilled in the art, such as the cytomegalovirus promoter. (CMV).
• CMV cytomegalovirus promoter.
• the expression vector may further contain other regulatory sequences corresponding to an origin of replication, a ribosome binding site, one or more splice sites, a polyadenylation site, or a transcription termination site.
• An expression vector that can be used in the process according to the invention may correspond, in a nonlimiting manner, to a YAC (artificial yeast chromosome), a BAC (artificial bacterial chromosome), a viral vector, a plasmid vector, a phagemid, a cosmid, an RNA vector, a vector derived from a baculovirus, a phage, a transposon or a RNA or DNA molecule, linear or circular.
• YAC artificial yeast chromosome
• BAC artificial bacterial chromosome
• viral vectors examples include retroviruses, adenoviruses, parvoviruses, coronaviruses, orthomyxoviruses, rhabdoviruses, paramyxoviruses, picornaviruses, alphaviruses, adenoviruses, herpesviruses and poxviruses.
• the expression vector used is a plasmid vector.
• Techniques for introducing into an egg or oocyte a molecule of nucleic acids are well known to those skilled in the art. Examples of such techniques include microinjection, electroporation, transfection with liposomes or modified lipids such as lipofectamine ® (IN VITROGEN), or with the aid of precipitated calcium phosphate. This introduction is preferably carried out by the microinjection technique.
• the targeted genomic modification introduced as a result of homologous recombination at the DNA double - strand break site may correspond to either a deletion of a genomic sequence, in the case where recombination takes place between two homologous sequences of the genomic sequence. the genomic DNA on either side of the break site, or an insertion in the case where the recombination takes place, at homologous regions, between an exogenous nucleic acid sequence and the genomic DNA.
• said method further comprises a step of introducing into the oocyte or egg an exogenous nucleic acid sequence which has homology with the nucleic acid sequences located upstream and downstream of the recognition site for the endonuclease which is present in genomic DNA.
• exogenous nucleic acid sequence is meant a double-stranded DNA sequence which is introduced into an egg or oocyte, which may be in linear or circular form.
• said exogenous nucleic acid sequence is in circular form.
• the concentration of the nucleic acid sequence administered per egg or oocyte is between 1 and 50ng per ⁇ l, preferably between 5 and 40 ng per ⁇ l, and particularly preferably between 10 and 30 ng per ⁇ l.
• volume injected per egg or oocyte is part of the general knowledge of those skilled in the art and is of the order of 10% of the volume thereof.
• the volume likely to be injected into a zebra or Medaka egg or oocyte is between 300 ⁇ l and 1 ⁇ l.
• the amount of nucleic acid introduced per egg or oocyte is then between 0.3 and 50 ⁇ g, preferably between 1.5 and 40 ⁇ g, and particularly preferably between 3 and 30 ⁇ g.
• said exogenous nucleic acid sequence is derived from the genomic sequence, in particular a mutated form of said genomic sequence or an isoform of said genomic sequence which is derived from another individual or from another organization.
• the homologous recombination mechanism results in an insertion of the exogenous nucleic acid sequence which appears to be a "replacement" of the genomic sequence.
• said exogenous nucleic acid sequence comprises a nucleic acid sequence of interest which is flanked by two distinct nucleic acid sequences, which exhibit homology with the nucleic acid sequences. nucleic acids located upstream and downstream respectively of the recognition site for the endonuclease that is present in the genomic DNA.
• the nucleic acid sequence of interest may correspond to a gene or regulatory sequence (such as a promoter or enhancer) whose activity and / or associated phenotype is to be determined in the transgenic vertebrate obtained by the method of the invention.
• the nucleic acid sequence of interest may comprise a reporter gene, such as beta-galactosidase, GFP (green fluorescent protein), RFP (red fluorescent protein), or a selection gene, such as neomycin phosphotransferase, at the same time. hygromycin phosphotransferase, histidinol dehydrogenase or thymidine kinase.
• the use of such a selection reporter gene may facilitate identification of eggs or oocytes with the targeted genomic modification desired.
• said reporter or selection gene does not include associated promoter sequences so as to identify the eggs or oocytes having the expected expression profile corresponding to the targeted targeted genomic modification.
• the nucleic acid sequence (s), which have a homology with the nucleic acid sequences located upstream and downstream of the recognition site for the endonuclease have a length of at least 50 base pairs. preferably at least 100 base pairs, and particularly preferably at least 250 base pairs.
• the said sequences may have a larger size, provided that size of more than 1000 base pair does not increase the efficiency of homologous recombination.
• said homologous nucleic acid sequence (s) has a sequence identity of at least 80% with the nucleic acid sequences located upstream and downstream of the recognition site for the endonuclease in the genomic DNA. preferably an identity of at least 90%, and particularly preferably an identity of at least 95%.
• the identity between two nucleic acid sequences corresponds to the percentage of identical nucleotides located at an identical position between two nucleic acid sequences.
• Numerous programs or algorithms make it possible to calculate percentages of identity among which FASTA or BLAST. These programs are available on NCBI (National Center for Biotechnology Information, http: // www. Ncbi .nlm. Nih. Gov /).
• the homology is determined by the BLAST program, and particularly preferably with a BLAST program using the BLOSUM62 matrix.
• said exogenous nucleic acid sequence is a vector.
• vector is meant a nucleic acid sequence capable of transporting a sequence of nucleic acids of interest to which it is linked.
• vectors that may be used in the process according to the invention, mention may be made, without limitation, of a YAC (artificial yeast chromosome), a BAC (artificial bacterial chromosome), a suitable viral vector, such as an adenovirus, a plasmid vector, a phagemid or a cosmid.
• the vector used is a plasmid vector.
• said exogenous nucleic acid sequence has no recognition site for the endonuclease.
• Techniques for introducing into an egg or oocyte a nucleic acid sequence are well known to those skilled in the art. Examples of such techniques include microinjection, electroporation, transfection with liposomes or modified lipids such as lipofectamine ® (IN VITROGEN), or with the aid of precipitated calcium phosphate. This introduction is preferably carried out by the microinjection technique.
• exogenous nucleic acid sequence may be delayed or simultaneous with respect to that of the endonuclease or the nucleic acid molecule allowing the expression of said endonuclease.
• the method according to the invention further comprises a step of culture of a previously fertilized oocyte or of the egg having a targeted genomic modification under conditions adapted to allow the development non-human vertebrate.
• the culture conditions used allow the long-term development of the non-human vertebrate.
• this culture step corresponds to the incubation of the eggs at a temperature of the order of 28 ° C., more or less 1 or 2 ° C.
• said method further comprises a step prior to the culturing step which corresponds to an incubation of the egg at a temperature of 5 to 20 ° C. below the temperature culture, preferably less than 10 to 15 ° C, and for a time to maintain egg viability greater than 5%, ie the number of eggs arriving until hatching, preferably higher 10%, and particularly preferably greater than 15%.
• the maximum time during which the eggs or oocytes can be maintained can be determined simply by those skilled in the art, and is a function of the temperature resistance of the eggs or oocytes used.
• such an incubation is carried out for a time of between 1 and 24 hours, preferably between 1 and 20 hours, and particularly preferably between 1 and 10 hours.
• this prior step corresponds to an incubation at a temperature of between 10 and 25 ° C., preferably between
• the step of identifying the eggs or oocytes presenting the desired targeted genomic modification is carried out on cells derived from the nonhuman vertebrate organism obtained during the development of said eggs or oocytes after fertilization, preferably on cells derived from of the mature nonhuman vertebrate organism.
• the Pa 1 TI-GFP-I construct was obtained by inserting, in the plasmid Pa 1 TI-EGFP (Goldman et al., Transgenic Res., Vol.10 (1), p: 21-33, 2001; HIEBER and al., J. Neurobiol., vol.37 (3), p: 429-440, 1998)), a recognition site for I-SceI meganuclease between the zebrafish ⁇ -tubulin promoter and the reporter gene of EGFP (Enhanced Green Fluorescent Protein).
• the Pa 1 TI-EGFP construct was digested with the BamHI enzyme (BIOLABS) and the digested construct was then purified.
• BIOLABS BamHI enzyme
• the BamHI-digested Pa 1 TI-EGFP construct was then dephosphorylated and then purified again.
• the pact-GFP12 construct was obtained as described in THERMES et al. (Mechanisms of Development, 118, p: 91-98, 2002).
• the Oc 1 TI-EGFP-I transgene in linearized form was obtained by digestion of the Pa 1 TI-EGFP-I construct with the XhoI and AfIII enzymes (BIOLABS).
• the XhoI-AflII fragment containing the transgene was then purified on a QIAEX II® column (QIAGEN) and then filtered on an Elutip-D® column (SCHLEICHER AND SCHUELL).
• the act-GFP12 transgene in linearized form was obtained by digestion of the pact-GFP12 construct with meganuclease I-Scel (ROCHE DIAGNOSTICS). The I-scel-I-Scel fragment containing the transgene was then purified as before.
• the transgene O 1 TI-EGFP-I was injected in linear form (XhoI-AfIII fragment) and the transgene act-GFPI2 in linear form (Iscel-I-Scel fragment) or circular (pact-GFP12). .
• the results showed an expression profile similar to that of the Pa 1 TI-EGFP construct in the egg.
• the expression profile of the act-GFP12 transgene is similar to that observed in THERMES et al. (2002, cited above).
• the 4 founding members obtained by the integration of the transgenic Ci 1 TI-EGFP-I were named FO .191, FO .251, FO .341 and FO .361.
• the F1 individuals from these founders had uniform green fluorescence in the CNS, which was observable early in neurogenesis, at the early-neural stage (25hpf, st.17).
• Fl individuals from this founder had several levels of GFP expressions.
• FO .25-1, FO .25-2 and FO .25-3 were named.
• F1 can be due to the segregation of different concatemers of the transgene integrated into the genome, in FO, in three sites. Genetically distinct integration. These embryos (F1) were bred until hatching and only weakly and moderately fluorescent eggs hatched and gave rise to sexually mature adult fish. Transmission of the transgene to subsequent generations (ie F2, F3 and F4) remained uniform, which confirms the stable integration of the transgene.
• the average transmission rate of founding individuals was estimated by following the expression of transgenes in the offspring of positive F1 individuals. The rates obtained were very variable and less than 50%. In the case of the transgene U 1 TI-EGFP-I, the average was 30 ⁇ 12, 5%. Only one of the four founding fish (FO .191) then had a significant 50% transmission, corresponding to the percentage of hemizygous transmission. In this case, it is likely that the transgene has integrated into FO at a single site and in all cells of the germ line. Ultimately, the results show a marked improvement in the efficiency of transgene integration into the germline in the presence of two or a single I-SceI site.
• Sacl digestion a site outside the region recognized by the probe
• the two bands of approximately 4kb probably correspond to transgene insertions in direct tandem and type I inverse tandem respectively (FIGURE 2A and 2B, respectively).
• the smaller bands (2kb and about 3kb) these most likely correspond to junction fragments between the integrated DNA and the genomic DNA.
• the intensity of the two 4kb bands, relative to the junction fragments indicates the presence of both types of tandems in large numbers in the genome and in comparable proportions.
• the two FO lines have an insertion profile with simple concatamers and low copy number (Type I inverse tandems).
• the transgenic lines obtained to carry out homologous recombination with meganuclease I-SceI it is important that the (s) I-Scel site (s) integrated into the genome itself (s) functional (s).
• the genomic DNA of these lines was purified. Said genomic DNA was then amplified by PCR using specific primers positioned on either side of the I - Sce I recognition site (FIG. 3A). The amplified DNA fragment has a length of 500 base pairs.
• the PCR products obtained were then digested with the enzyme I-Scel (ROCHE DIAGNOSTICS) according to the manufacturer's instructions, and finally deposited on an electrophoresis gel. The results are shown in Figure 3B.
• Gel migration of the reaction products reveals the presence of a band at about 500 bp, corresponding to the uncut DNA, and two other smaller bands 200 and 300 bp (cut DNA, FIGURE 3A and 3B). .
• the presence of the 500bp band may result from incomplete enzymatic digestion or the presence of mutated I-Scel sites. In all cases, the results show that the 4 lines analyzed have non-mutated functional I-Scel sites, which are variable in number depending on the line analyzed.
• EXAMPLE 2 Targeted insertion of a transgene into the genome of a transgene line of medaka with an I-SceI site
• transgene In order to integrate a transgene into the medaka genome in a targeted way, we tested the gap repair technique. For this, we used a second transgene containing the tracer gene of InRFP 1 (monomeric red fluorescent protein) surrounded on both sides by sequences of at least 500 bp perfectly homologous to the regions surrounding the I-SceI site of transgene (X 1 TI-EGFP-I (CR, Repair Construction) . The 5 'homologous region corresponds to the intronic sequence of the Cx 1 TI promoter, which rules out any possibility of HiRPF 1 expression in episomal form.
• InRFP 1 monomeric red fluorescent protein
• SacI-NotI fragment of plasmid P 1 TI-EGFP (1.7 kb), corresponding to the regions of homology located on either side of the I-SceI site, was purified and cloned into the vector pCRII -TOPO® (Invitrogen) linearized by a SacI-NotI digestion.
• the resulting RH plasmid was controlled by sequencing.
• the resulting construct served as template for amplifying the InRFP 1 -PoIyA sequence by PCR by adding a BgIII site at each end (sense primer (SEQ ID NO: 3): 5 '- GAAGATCTCTTAAGCATGGCCTCCTCCGAGGAC-3'; antisense primer (SEQ ID NO: 4): 5'-CCTAGATCTGCTAGCATACATTGATGAGTTTGGAC-3 ').
• the resulting PCR product was cloned into plasmid pCRII-TOPO® (Invitrogen) and the sequence was monitored by sequencing.
• the InRFP 1 -PoIyA fragment was then isolated by performing SgIII digestion of this construct.
• repair construct was generated by introducing the InRFP 1 -PoIyA fragment (BgIII digestion) at the BamHI site of the RH construct.
• a Pa 1 TI-InRFP1-EGFP control construct was generated by introducing this same InRFP 1 -PoIyA fragment into the P 1 TI-EGFP construct.
• the repair construct was co-injected in circular form with meganase I-SceI in a stage one medaka egg according to the described protocol. in Thermes et al. (2002, cited above).
• the eggs used were from transgenic fish lines (F3, FO25-1 and -2, FO19 and FO36).
• the repair construct (CR) was injected in circular form at a final concentration of approximately 10 ng / ⁇ l, after amplification with a Midiprep® kit (Qiagen) and filtration (0.2 ⁇ m filter), in the presence of I-SceI at the concentration of 1 unit per ⁇ l.
• the eggs After injecting the stage one cell medaka eggs, the eggs are placed in an incubator at 28 ° C until hatching.
• the construct controls Pa 1 TI-InRFP1-EGFP was injected alone into a one-celled medaka egg according to the same protocol. The injection of this one led to a good expression of the InRFP 1 in the embryo, without traces of green fluorescence.
• the injected embryos were observed with a magnifying glass for their green and red fluorescence (in particular), towards the stages 28 (30 somites, 64 hpf) and 32 (end of somitogenesis,
• Random integrations are first carried out according to the protocol described in Example 1, but with meganucleases I-CreI and I-Ceul and constructs respectively comprising a recognition site for meganuclease I-CreI (SEQ ID NO: 5;

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