Method for preparing humanized ACE2 mouse model
A mouse model, humanized technology, applied in the field of biomedicine
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Embodiment 1
[0038] Cloning of human ACE2 coding sequence (hACE2 CDS)
[0039] Using RNA derived from human tumor cell line K549, use poly-dT primers to bind to poly-A at the 3' end of the RNA, plus Super IV reverse transcriptase Prepare cDNA library. According to the human ACE2 gene sequence (NM_021804.3) provided by NCBI, primers were designed for the coding sequence, the upstream primer hACE2-Fwd=SEQ ID NO.7 and the downstream primer hACE2-Rev=SEQ ID NO.8, using the above cDNA library as a template , human ACE2 coding sequence primers and DNA polymerase ( II Fusion DNA Polymerase, Agilent) for PCR specific amplification of ACE2 coding sequence. The amplified ACE2 coding sequence was subjected to agar gel electrophoresis, recovered and purified, cloned into a pHE (Tools Biotech.) vector, and the ACE2 coding sequence was sequenced using the T7 promoter and reverse sequencing sequence on the vector. Select the correct and mutation-free ACE2 coding sequence for DNA cloning, cut off t...
Embodiment 2
[0041] Preparation of targeting sgRNA, ssODN and Cas9 RNP
[0042] According to the mouse ACE2 genome sequence provided by NCBI (NC_000086.7:164139342-164188418), design sgRNA sequences for exon 2 and exon 15 sequences, synthesize upstream and downstream sgRNA primers, and add T7 promoter at the 5' end of the positive strand sequence Sequence; The primer sequence T7-MmAce2_Ex2=SEQ ID NO.3 of upstream sgRNA, the primer sequence T7-MmAce2_Ex15=SEQ ID NO.4 of downstream sgRNA; Use The kit (New England BioLabs) contains T7 RNA polymerase for in vitro transcription, synthesizes and purifies sgRNA to a concentration of about 1000ng / ul, and stores it at -80°C for future use. The two ssODNs contain the homologous sequence of mouse ACE2 exon 2 and the 5' end of the human ACE2 coding region, and the 3' end of the human ACE2 coding region and the mouse ACE2 exon 15 homologous sequence, which are chemically synthesized. The sequence of the upstream ssODN is ssODN -UP=SEQ ID NO.5, sequen...
Embodiment 3
[0044] The preparation, culture and transplantation of fertilized mouse embryos, the process is as follows image 3 Shown in B.
[0045] C57BL6 female mice were given 5IU of PMSG, and 48 hours later were given 5IU of hCG and mated with C57BL6 male mice. After 16 hours, the vaginal suppository of the female mice was checked. After the successful mating female mice were euthanized, the oviducts on both sides were removed to obtain fertilized eggs. Fertilized eggs were incubated with hyaluronic acid (Hyaluronidase, 0.1%) at room temperature for 3 minutes to remove cumulus cells, and transferred to M2 operating solution (M5910, Sigma-Aldrich) for use. Mouse embryos after pronuclear injection of the protein-nucleic acid complex prepared in Example 2 were placed in KSOM culture medium, covered with mineral oil (M8410, Sigma-Aldrich) and placed in 100% humidity, and cultured in a 5% CO2 environment for 72 hours until blastocysts Expect. The ICR female mice that were sham-mated wit...
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