Construction and application of severe combined immunodeficiency animal model
An animal model, immunodeficiency technology, applied in the direction of using microinjection, recombinant DNA technology, other methods of inserting foreign genetic materials, etc., can solve problems such as affecting the replication effect of animal models, and achieve reliable knockout efficiency and immunodeficiency. High, good effect of tumor cell growth
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Embodiment 1
[0036] Example 1 Oligonucleotide Synthesis and Plasmid Construction
[0037]According to the Rag2 and Il2rg gene sequences reported by Genbank, sgRNAs with targeting effects were designed for the second exon of Rag2 gene and the first exon of Il2rg gene, respectively. The sgRNA sequences are shown in Table 1. After the sgRNA is synthesized, it is annealed and ligated into the pX330 vector to construct the pX330 plasmid containing the sgRNA. Plasmid extraction and sequencing showed that the plasmid was constructed correctly. After the sgRNA and Cas9 were cloned, they were transcribed in vitro under the action of the T7 promoter. The transcript was identified by electrophoresis as a single band with OD260 / 280>1.9 and OD260 / 230>2.3.
[0038] Table 1 sgRNA targets and oligonucleotide sequences
[0039] sgRNA name
Embodiment 2
[0040] Example 2 In vitro transcription, mRNA microinjection and fertilized egg cell transplantation
[0041] Using pX330-Rag2-sgRNA and pX330-Il2rg-sgRNA plasmids as templates, Rag2-sgRNA and Il2rg-sgRNA containing T7 promoter were cloned by PCR. The Cas9 gene was cloned in the same way, recovered after purification, and the DNA was dissolved in DEPC water for preservation. 200ng of Rag2-sgRNA, Il2rg-sgRNA and Cas9 purified products were taken for in vitro transcription, and stored at -80°C after quantification.
[0042] Male and female mice of 8-week-old BALB / c mice were co-caged one day before use, and the female mice with plugs were dissected 24 hours later, and fertilized eggs were obtained from the fallopian tubes. The transcribed 20 μg / μL Rag2-sgRNA, 10 μg / μL112rg-sgRNA and 5 μg / μL Cas9 mRNA were mixed and injected into the cytoplasm of fertilized egg cells with Eppendorf NK2 microinjector. The fertilized egg cells were further cultured in a 5% CO2 incubator at 37°C f...
Embodiment 3
[0043] Embodiment 3: establishment and genotype identification of gene knockout mouse strain
[0044] The tail tissues of the F0 generation mice born about 2 weeks old were taken out, and after lysing, the genomic DNA of the mice was extracted with a kit, and the Rag2 and Il2rg genes of the F0 generation mice were cloned by PCR. The PCR reaction conditions were: pre-denaturation at 94°C for 3min, denaturation at 94°C for 30s, annealing at 60°C for 40s, extension at 65°C for 40s, and a total of 35 cycles. After cycling, extend at 65°C for 6 min. Take 10 μL of the PCR product for electrophoresis, and sequence it after the size is correct. Mutated F0 generation mice were mated with wild-type mice to obtain F1 generation heterozygous mice, and F1 generation heterozygous mice were crossed to obtain double-gene mutant homozygous mice. A total of 740 BALB / c mouse embryos were collected Among them, 609 were fertilized egg cells, 249 of which survived after microinjection, and were t...
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