sgRNA targeting the apobec3g gene and a method for knocking out the apobec3g gene in cynomolgus monkeys

A gene knockout, cynomolgus monkey technology, applied in the direction of DNA / RNA fragments, other methods of inserting foreign genetic materials, genetic engineering, etc.

Active Publication Date: 2021-08-24
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • sgRNA targeting the apobec3g gene and a method for knocking out the apobec3g gene in cynomolgus monkeys
  • sgRNA targeting the apobec3g gene and a method for knocking out the apobec3g gene in cynomolgus monkeys
  • sgRNA targeting the apobec3g gene and a method for knocking out the apobec3g gene in cynomolgus monkeys

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 APOBEC3G Gene Target Efficiency Detection

[0057] (1) Design target

[0058] 针对食蟹猴APOBEC3G基因(基因ID为NC_022281.1)的mRNA确立靶序列;根据食蟹猴APOBEC3G基因的mRNA序列(cagcgatct cccagtgagc cccagaaggg gtcagaggggcaggtgtgga ggctccagca agtgagcggg agccccttct gacaggtgct aagggatgtg gggagcaaggggaaggaggg tggggtgaaa gggaggaagc gtggagaggg agggggaggc agggcagacc acgacgagggctcttactcc tctgggcttt ttcccccgct gtccacagac atttgatgga tccaggcacg ttcacttccaactttaacaa taaaccttgg gtcagtggac agcatgagac ttacctgtgt tacaaggtgg agcgcctgcacaatgacacc tgggtcccgc tgaaccagca caggggcttt ctacgcaacc aggtgaccaa cccagccacccacatccagg cagggctctc ccaatccagg gacacgcatg ggcagaaggt tctgggcggt acctgtggtgtcccgcagag tgtctgtcac ctgtgcttcc tgcagctgct gctgcttggc cctggggttg gggggggaaactttggcttc agtgactctc),设计15个靶序列,其中3个靶序列的核苷酸序列如下所示:

[0059] sgRNA1: 5'-CAATAAACCTTGGGTCAG(TGG)-3';

[0060] sgRNA2: 5'-AGCGCCTGCACAATGACACCTGG(AGG)-3';

[0061] sgRNA3: 5'-TCCCGCTGAACCAGCACA(GGG)-3';

[0062] sgRNA5: 5'-GGAGGCAGGGCAGACCACG(AGG)-3';

...

Embodiment 2

[0086] Example 2 Cynomolgus monkey embryonic fibroblasts with APOBEC3G knockout

[0087] (1) Construction of knockout plasmid vector

[0088] After target in vitro activity detection, the present invention designs and synthesizes sgRNA primer oligo according to sgRNA1 and sgRNA3 targeting APOBEC3G gene (both located in the sixth exon of APOBEC3G gene), wherein the sequence of sgRNA primer oligo is as follows:

[0089] sgRNA1-Sense: 5'-AAACACCG-CAATAAACCTTGGGTCAG;

[0090] sgRNA1-Anti: 5'-CTCTAAAAC-CTGACCCAAGGTTTATTG;

[0091] sgRNA3-Sense: 5'-AAACACCG-TCCCGCTGAACCAGCACA;

[0092] sgRNA3-Anti: 5'-CTCTAAAAC-TGTGCTGGTTCAGCGGGA;

[0093] The above-mentioned sgRNA primer oligo was annealed to form an oligo dimer, which was combined with the vector backbone pCAG-T7-Cas9 (Miaoling plasmid platform addgene) ( figure 2 ) ligation, and let stand at 25°C for 5 minutes to insert the oligo dimer into the vector to obtain a knockout vector;

[0094] (6) Co-transfection of cynomolgus e...

Embodiment 3

[0106] (1) In vitro transcription and synthesis of sgRNA

[0107] ① Design and synthesize primers according to Example 1 target sgRNA1 and sgRNA3, wherein the nucleotide sequences of the primers are as follows:

[0108] T7-gRNA-F: 5'-TAATACGACTCACTATAGNNNNNNNNNNNNNNNNNNNNNN-3';

[0109] gRNA-PCR-R: 5'-AGCACCGACTCGGTGCCACTT-3';

[0110] Using the px459 vector as a template, T7-gRNA-F and gRNA-PCR-R as amplification primers, perform PCR amplification to obtain a transcriptional DNA template, wherein the PCR amplification system is the same as in Table 1;

[0111] (2) The transcribed DNA template prepared in step (1) was transcribed (Beijing Weishang Lide T7 Transcription Kit) and purified to obtain sgRNA, wherein the transcription system was the same as in Table 2;

[0112] (3) Development of embryos after vector injection

[0113] sgRNA3: 50ng / μL (final concentration after mixing) and Cas9mRNA (commercially available) 80ng / μL (final concentration after mixing) were mixed and...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to an sgRNA targeting the APOBEC3G gene and a method for knocking out the APOBEC3G gene in cynomolgus monkeys. The present invention establishes a target sequence for mRNA of cynomolgus monkey APOBEC3G gene and synthesizes corresponding sgRNA, then constructs a knockout vector, co-transfects cynomolgus monkey embryonic fibroblasts, and obtains cynomolgus monkey embryonic fibroblasts with knockout of APOBEC3G gene, Or mixed injection of sgRNA and Cas9 mRNA into cynomolgus monkey embryos to obtain APOBEC3G knockout cynomolgus monkey embryos. The invention can effectively realize gene knockout of APOBEC3G gene on cynomolgus monkey cells and cynomolgus monkey embryos through detection and identification of knockout effect, and lays a solid foundation for establishing AIDS animal models.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an sgRNA targeting the APOBEC3G gene and a method for knocking out the APOBEC3G gene in cynomolgus monkeys. Background technique [0002] Acquired immunodeficiency syndrome (Acquired immunodeficiency syndrome, AIDS) is a disease caused by human immunodeficiency virus (Human immunodeficiency virus, HIV). The virus targets human CD4 T lymphocytes, resulting in the loss of immune function of the infected person. Animal disease models are an indispensable tool for disease research. In AIDS disease models, although immunodeficiency viruses that can infect different species of animals have been found in other animals, none of the disease models can achieve HIV-1 virus infection and Show clinical manifestations of AIDS patients. The researchers hope that creating a non-human primate disease model of cynomolgus monkeys infected with HIV through gene knockout technology will ope...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/90C12N9/22
CPCC12N9/22C12N15/113C12N15/907C12N2310/10
Inventor 杨世华方志浩邓英华
Owner SOUTH CHINA AGRI UNIV
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