Construction method and application of hACE2 humanized transgenic pig
A humanized and genetic technology, applied in the field of genetic engineering, can solve the problems of high cost of feeding and breeding, limited wide application, difficult experimental operation, etc., and achieve the effect of simple design and high gene knock-in efficiency.
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Embodiment 1
[0075] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions. Example 1 Construction method of humanized ACE2 pig model
[0076] The technical process for the preparation of the humanized ACE2 pig model in the present invention mainly includes the following three aspects:
[0077] First, porcine ACE2 targeting and human ACE2 insertion strategy ( figure 1 )
[0078] Design sgRNA near the first exon ATG of the porcine ACE2 gene, use Cas9 nuclease to cut the target site, and then introduce a homologous repair vector containing hACE2 cDNA-polyA, taking about 1kb upstream of the start ...
Embodiment 2
[0103] Example 2 Detection of humanized ACE2 pig model for susceptibility to SARS-CoV-2
[0104] In the present invention, the humanized ACE2 pig model mainly includes the following three aspects for the detection of the susceptibility of SARS-CoV-2:
[0105] First, isolate and culture primary lung and kidney epithelial cells from the ACE2 humanized pig model and littermate wild-type control pigs.
[0106] The specific operation is as follows: kidney and lung tissues of hACE2 knock-in piglets and littermate wild-type control pigs were collected, washed with PBS, and then digested with 200 U / mL type I collagenase at 37° C. for 15 minutes. The primary epithelial cell culture medium is 15% fetal bovine serum, 1% penicillin and streptomycin, and 10 ng / μL epithelial growth factor added to DMEM basal medium. 2 cultivated under conditions.
[0107] Second, to observe the cytopathic effect of primary epithelial cells after SARS-CoV-2 infection ( Figure 8 ).
[0108]The specific o...
Embodiment 3
[0113] Example 3 Optimization experiment of sgRNA
[0114] The present invention designs 9 sgRNAs near the start codon ATG of the first exon of the pig ACE2 gene site, and tests the gene targeting efficiency of these sgRNAs in IBRS2 cells, each sgRNA sequence and the targeting efficiency of the pig ACE2 gene As shown in Table 1:
[0115] Table 1
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[0117]
[0118] It can be seen from Table 1 that in IBRS2 cells, the sgRNA3 gene targeting efficiency of the porcine ACE2 site is the highest, so it was selected as the target site used in the subsequent CRISPR / Cas9-mediated exogenous human ACE2 gene insertion system.
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