A kind of anti-mouse hepatitis virus mouse and preparation method thereof
A mouse hepatitis virus and mouse technology, applied in biochemical equipment and methods, other methods of inserting foreign genetic materials, animal husbandry, etc., can solve problems affecting the quality of experimental mice and experimental results, limited breeding environment, etc.
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Embodiment 1
[0057] Example 1 Construction and Activity Identification of CEACAM1a Knockout Vector pX330-Cas9-sgRNA Plasmid
[0058] 1. Determination of sgRNA target sites
[0059] 1. Experimental method
[0060] First obtain the genome sequence of Ceacam1 (Gene ID 26365), and find the start codon and exon region of the gene, according to the basic principles of designing sgRNA, through http: / / crispr.mit.edu / Find potential high-efficiency sgRNA sites.
[0061] 2. Experimental results
[0062] The following sgRNA was obtained:
[0063] sgRNA1: 5'-TGAAAACTATCGTCGTACTCAGG-3' (SEQ ID NO: 1);
[0064] sgRNA2: 5'-CATTGAGGCTGTGCCGCCCCAGG-3' (SEQ ID NO: 4);
[0065] sgRNA3: 5'-GCAAAGGCTCCAAGCGCCAGGGG-3' (SEQ ID NO: 7).
[0066] The three sgRNA sites are all located in the second exon of Ceacam1, and the specific targets of sgRNA1 are as follows: figure 1 shown.
[0067] 2. Construction of plasmids capable of simultaneously expressing Cas9 and sgRNA
[0068] 1. Experimental method
[00...
Embodiment 2
[0107] Example 2 Construction of CEACAM1a knockout mice
[0108] 1. The cultivation of experimental animals
[0109] Select 3-4 week old C57BL / 6J female mice, perform superovulation treatment (in order to obtain enough eggs), and mate them with 7-8 week old C57BL / 6J male mice to obtain embryos for microinjection . The vas deferens of CD1 male mice over 8 weeks old were mated with CD1 female mice over 6 weeks old to obtain pseudopregnant female mice. All experimental mice were multiplied in the Experimental Animal Center of Sun Yat-sen University.
[0110] 2. Microinjection and Embryo Transfer
[0111] The sgRNA1 and Cas9 mRNA obtained in the above steps are co-microinjected into embryos at the single-cell stage, since the sgRNA will guide the Cas9 protein to cut at the specific DNA site complementary to its sequence, and cause DNA double strand breaks (double strands breaks, DSBs), and then stimulate the cell's non-homologous end joining repair mechanism, resulting in the ...
Embodiment 3
[0140] Example 3 MHV-A59 Infects CEACAM1a Knockout Mice
[0141] 1. Experimental method
[0142] To further validate Ceacam1 - / - Whether mice can resist MHV infection, the present invention uses MHV-A59 strain to infect Ceacam1 respectively - / - mice and wild-type mice.
[0143] After the MHV-A59 standard strain proliferates in large quantities through L929 cells, determine its TCID50=10 5.7 After 1:500 dilution, each mouse was intraperitoneally injected with 0.5ml.
[0144] IgG detection: Blood collection and liver collection were carried out on days 3, 7, 14, 21, and 28 respectively (each sampling Ceacam1 - / - There were 5 mice and 5 wild-type mice each, and 1 wild-type mouse died of infection on the 28th day), and the IgG in the serum was detected according to the ELISA method of the national standard of the People's Republic of China (GB / T 14926.50-2001) . The OD value was measured with a microplate reader at a wavelength of 405 nm.
[0145] Pathological picture: mous...
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