Construction method and application of immunodeficient mouse animal model
A technology of immunodeficiency mice and animal models, which is applied in the field of animal genetic engineering and genetic modification, can solve time-consuming and serious problems, and achieve the effect of promoting basic research
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Embodiment 1
[0028] Construction of CRISPR-Cas9 expression system for Il2rg gene
[0029] Using CRISPR / Cas9 technology to establish Il2rg gene knockout mice, figure 1 Middle, A. Targeting sequence design for exon 1 of Il2rg gene. B. Generated base deletion and insertion mutations.
[0030] 1. According to the mouse Il2rg gene sequence, the sgRNA was designed and the sequence information of the sgRNA was obtained. The DNA sequence of the sgRNA specifically targeting the first exon of the Il2rg gene is shown in SEQ ID NO.1, that is, GAGGGCAGGGTGGAGCTCCA. Wherein SEQ ID NO.1 targets SEQ ID NO.2 (SEQ ID NO.2 is a part of Il2rg gene). Synthesize positive and negative single-strand Seq ID No.3 and Seq ID No.4 respectively, and then anneal to form SEQ ID NO.1.
[0031] 2. Construction of sgRNA vector plasmid:
[0032] (1) Design and synthesize the DNA sequence of the sgRNA recognition region that recognizes the first exon of the Il2rg gene, namely Seq ID No.3 and Seq ID No.4, as shown in Tab...
Embodiment 2
[0042] Example 2 In vitro transcription
[0043]Use T7-Cas9 PCR and T7-sgRNA PCR products to carry out in vitro transcription mediated by T7 promoter, that is, use T7 promoter as the promoter of in vitro transcription, and use RNA polymerase to realize the transcription process from DNA to mRNA in vitro, specifically The method is as follows: use the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies) to transcribe the T7-Cas9 PCR product in vitro, and use the MEGAshortscript T7 kit (Life Technologies) to transcribe the T7-sgRNA PCR product in vitro. The mRNA produced by transcription was purified. The specific method was as follows: Cas9 mRNA was purified by MEGAclear kit (Life Technologies), sgRNAs were purified by ethanol precipitation, mRNA was dissolved in pure water, and the concentration of purified mRNA was measured by spectrophotometer.
[0044] 7-Cas9 PCR primers are shown in Table 4, T7-sgRNA PCR primers are shown in Table 5, gRNA-Il2rg-F is the forward primer for SE...
Embodiment 3
[0054] Example 3 Production of gene-targeted mice using CRISPR-Cas9 system mRNA for Il2rg gene
[0055] 1. Pronuclear injection and embryo transfer
[0056] The pronuclear fertilized eggs of mice were taken, and the premixed Cas9mRNA / sgRNA mixture (the final concentration of Cas9mRNA was 100ng / μl, and the final concentration of sgRNA was 50ng / μl) was injected into the cytoplasm of the mouse fertilized eggs using a microinjector, and then Transplant into the oviduct of the recipient mother mouse to produce gene-targeted mice, and the injection volume is 0.5-1ul.
[0057] 2. Identification of gene targeting mice
[0058] After the birth of the surrogate mother mice, cut off about 1 cm of the mouse tail when the offspring grow to 2 weeks old, digest with proteinase K at 55°C, and extract the genome DNA of the mouse tail with phenolic extraction. Using mouse genomic DNA as a template, primers targeting exon 1 of the Il2rg gene were designed, amplified, and the obtained PCR produ...
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