Compositions and methods for producing genetically modified animals
a technology of genetically modified animals and compositions, applied in the field of compositions and methods for producing genetically modified animals, can solve the problems of unpredictable contribution of germ line es cells in chimeras, and achieve the effect of enhancing the production of first litter offspring and endogenous
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example 1
Generation of Conditional GILZ (Tsc22d3) Knockout Mice
[0188]A targeting vector was constructed to flank exon 4 (ENSMUSE00000815383) of the mouse Tsc22d3 (ENSMUSG00000031431) gene with loxP sites via homologous recombination. Cre-recombinase mediated recombination of the loxP sites leads to the deletion of exon 4 (e.g., of transcript Tsc22d3-006 with the following Vega accession No. OTTMUST00000045354). The CDS of exon 4 codes for the complete sequence of the TSC22 (PF01166) domain. A schematic overview of the targeting vector is shown in FIG. 1.
[0189]A neomycin selection cassette (neo) for selection in ES cells was inserted downstream of exon 4. The selection cassette was flanked with FRT sites to enable removal by FLP-mediated recombination. Individual loxP sites were inserted upstream of exon 4 and downstream of the selection cassette. The 5′ and 3 homology arms of the vector were approximately 8.0 kb and 6.0 kb, respectively.
[0190]The linearized targeting vector was electroporate...
example 2
Generation of Targeted Mice Using Female Tsc22d3 Conditional Knockout Mice as Blastocyst Donors
[0191]21 to 25 day old Tsc22d3 conditional knockout female mice on a C57BL / 6 background are injected with pregnant mare serum. Two days later the mice are injected with human chorionic gonadotropin and mated for 24 h to C57BL / 6 Cre-recombinase males. Six days after the first injection blastocysts are extracted from the Tsc22d3 conditional knockout females. These blastocysts are used as recipients for microinjection of targeted BALB / c ES-cells and the microinjected blastocysts are transferred into pseudopregnant CBB6F1 foster females. The resulting chimeras are crossed to BALB / c females. Male chimeras with testis derived from blastocyst cells are expected to be sterile.
example 3
Generation of Conditional Knockout Mice as Blastocyst Donors
[0192]Conditional Knockout of a fertility gene ROSA26 Allele Variant A contains a nucleotide sequence that codes for a disruptor molecule (e.g., shRNA which has a transcript of a fertility gene as target, antibody directed against a protein that is encoded by a fertility gene, etc.). A fluxed Stop cassette inhibits the expression of the disruptor molecule. An illustrative targeting vector for making a targeted ROSA26 Allele A is shown in FIG. 2.
[0193]ROSA26 Allele Variant B contains the CDS for a Cre recombinase. An non-limiting example of a targeting vector for making a targeted ROSA26 Allele Bis shown in FIG. 3.
[0194]Breeding partner one is homozygous for the ROSA26 Allele Variant A.
[0195]Breeding partner two is homozygous for the ROSA26 Allele Variant B.
[0196]Breeding partner one can be male or female. Breeding partner two vice versa.
[0197]The offspring from crossing Breeding partner one with Breeding partner two will re...
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