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.

Pending Publication Date: 2021-10-22
BEIJING DHELIXON BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, problems such as slow reproduction rate, high feeding and breeding costs, difficult experimental operation and ethical reasons of non-human primate models further limit their wide application (Takayama, 2020; Abdel-Moneim and Abdelwhab, 2020; Pandey et al., 2020)

Method used

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  • Construction method and application of hACE2 humanized transgenic pig
  • Construction method and application of hACE2 humanized transgenic pig
  • Construction method and application of hACE2 humanized transgenic pig

Examples

Experimental program
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Effect test

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

[0116]

[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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Abstract

The invention discloses a construction method and application of an hACE2 humanized transgenic pig. A CRISPR-Cas9 gene editing technology is adopted, hACE2cDNA is used for replacing an ACE2 gene of a pig, and the problem that a pig model cannot well reproduce human disease characteristics due to the fact that pig endogenous ACE2 is also expressed while humanized ACE2 is expressed is solved. The hACE2 humanized pig model constructed by the invention is an ideal large animal pig model capable of simulating clinical symptoms and treatment response of human COVID-19, can be used for research on SARS-CoV-2 infection and pathogenesis, and is particularly suitable for research on multi-organ diseases, severe diseases, chronic diseases and sequelae caused by SARS-CoV-2 infection, the model can be used for development and testing of diagnostic technology, antiviral therapy and vaccines and evaluation of curative effect and safety of candidate drugs, and is beneficial to solving the problem of lack of an effective new coronal pneumonia animal model at present.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a construction method and application of hACE2 humanized transgenic pigs. Background technique [0002] The novel coronavirus (also known as Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) is a newly discovered virus since 2019 that can cause viral pneumonia / lung infection in humans. Study the mechanism of SARS-CoV-2 infection and pathogenesis, develop and test diagnostic techniques, antiviral therapies and vaccines against the virus, simulate its clinical symptoms and treatment responses, evaluate the efficacy and safety of candidate drugs, determine Biomarkers related to the degree and course of the disease require a large number of animal models similar to human clinical pathogenesis. [0003] Patients with new coronary pneumonia show a wide range of disease symptoms from asymptomatic to severe pneumonia and even death (Wu and McGoogan, 20...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/113C12N15/90C12N15/877C12N9/22C12N15/55C12N5/10A01K67/027
CPCC12N15/8509C12N15/1137C12N15/907C12N15/8775C12N9/485C12N9/22A01K67/0276A01K67/0278C12Y304/17023C12N2310/20A01K2207/15A01K2217/072A01K2217/075A01K2227/108A01K2267/0337Y02A50/30
Inventor 吴森杜旭光郭紫航
Owner BEIJING DHELIXON BIOTECHNOLOGY CO LTD
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