Composition, application, cell and gene-editing pig preparation method for modifying amino acid 561 of CD163 gene

A CD163, gene editing technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, botanical equipment and methods, etc. Strong and wide-ranging effect

Pending Publication Date: 2019-11-12
中农种源(深圳)科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in addition to playing an important role in mediating PRRSV invasion, CD163 also acts as a hemoglobin scavenger receptor to mediate the absorption of hemoglobin, so directly knocking out CD163 may affect other physiological functions of the body

Method used

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  • Composition, application, cell and gene-editing pig preparation method for modifying amino acid 561 of CD163 gene
  • Composition, application, cell and gene-editing pig preparation method for modifying amino acid 561 of CD163 gene
  • Composition, application, cell and gene-editing pig preparation method for modifying amino acid 561 of CD163 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Construction of recombinant plasmid pX330-CD163-gRNA vector and single-stranded donor sequence design

[0088] 1. First lock the sequence around the 561st amino acid of the porcine CD163 gene, use the website developed by MIT Zhang Feng Laboratory (http: / / crispr.mit.edu) to score the gRNA, and select from the candidate gRNA The sequence of a gRNA close to the R561 site and having a higher score is shown in SEQ ID NO: 1 (CD163-gRNA sequence: GCTACATGTCCCGTCAGGGC). The complementary paired oligonucleotide sequences shown in SEQ ID NO: 3 (CD163-gRNA-F sequence: caccGCTACATGTCCCGTCAGGGC) and SEQ ID NO: 4 (CD163-gRNA-R sequence: aaacGCCCTGACGGGACATGTAGC) were synthesized according to the gRNA sequence. 同时,根据gRNA序列本实施例还设计了一条如SEQ ID NO:2(CD163-ssODN序列:GACAGATCTGGGCTGAAGAATTCCAGTGTGAGGGGCACGAGTCCCACCTTTCACTCTGCCCAGTAGCGCCGGCCCCTGACGGGACATGTAGCCACAGCAGGGACGTCGGCGTAGTCTGCTCAAGTGAGACCCAGGGAATGTGTTCACTTT)所示的ssODN序列作为单链donor序列,当单链donor序列替换掉野生型序列后,R561 It was successfully r...

Embodiment 2

[0090] Example 2 Establishment of Monoclonal Porcine Fibroblasts Precisely Modified at the 561st Amino Acid of CD163 Gene

[0091] 1. Preparation of Porcine Fetal Fibroblasts

[0092] Remove the head, tail, limbs, viscera and bones from the 35-day-old pig embryo, and clean up the blood. Continue to cut the fetus with elbow ophthalmic scissors for 30 minutes to ensure that it is fully shredded. Pipette the shredded fetal tissue into a 15mL centrifuge tube with the blue tip of the scissors, add 5mL of complete medium, and remove the above solution after natural sedimentation for several minutes. And add a few drops of fetal calf serum to the lower tissue block, suck it out with a 15cm glass pasteur tube bent at the tip of 1cm, spread it in two T75 culture flasks, place the bottom of the bottle upward, and add 15mL full culture to the opposite side After 6-8 hours, carefully turn over the culture bottle, immerse the tissue pieces in the culture solution, change the solution ever...

Embodiment 3

[0101] Example 3 Preparation of gene-edited pigs with precise modification of the 561st amino acid of CD163 gene by somatic cell nuclear transfer technology

[0102] The homozygous knockout positive cells obtained in Example 2 were used as nuclear transfer donor cells, and the young pig oocytes matured in vitro for 40 hours were used as nuclear transfer recipient cells, and the nuclear transfer donor cells were transferred into the enucleated oocytes After electric fusion and activation, recombinant cloned embryos were constructed, and the cloned recombinant embryos with good development were selected and transferred into the uterus of natural estrous large white sows for pregnancy. The surgical embryo transfer step was ventilator anesthesia, and Maintain anesthesia with 2% chloral hydrate, lie supine on the operating rack, make a surgical incision about 10 cm long in the midline of the abdomen, expose the ovaries, fallopian tubes and uterus, use the embryo transfer glass tube ...

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Abstract

The invention provides a composition, application, cell and gene-editing pig preparation method for modifying an amino acid 561 of a CD163 gene, and relates to the technical field of biology. The composition for modifying the amino acid 561 of the CD163 gene comprises recombinant plasmids and single-stranded donor sequences. The recombinant plasmids comprise gene-editing vector skeletons and a section of DNA sequences connected to the vector skeletons. The single-stranded donor sequences are single-stranded DNA sequences in which the amino acid 561 of the CD163 gene is precisely replaced, theamino acid 561of the CD163 gene is precisely modified, and meanwhile, the situation that the normal expression of remaining amino acids of CD163 gene is destroyed or changed can be avoided; and therefore, on the basis of resisting PRRSV infection, the physiological activity function of CD163 protein is retained to the greatest extent, and the advantages of wide application range and high gene-editing efficiency are achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a composition for modifying the 561st amino acid of the CD163 gene, an application, a cell and a method for preparing a gene-edited pig. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) has the characteristics of high infectivity and strong lethality, and is considered to be one of the most urgent porcine infectious diseases to be overcome. Statistics show that the annual economic loss caused by PRRS in the United States and Europe is as high as 644 million US dollars and 600 million euros. [0003] CD163 protein is considered to be the key receptor for PRRSV (porcine reproductive and respiratory syndrome virus) to enter cells. Since 2014, several units have successfully produced CD163 gene knockout pigs, and challenge experiments have confirmed that CD163 gene knockout can completely resist PRRSV infection. However, in addition to playing an impor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/12C12N5/10A01K67/027
CPCA01K67/0275A01K2217/072A01K2227/108A01K2267/02C07K14/70596C12N5/0603C12N5/0656C12N15/85C12N15/8509C12N15/907C12N2510/00C12N2800/107
Inventor 李奎牟玉莲徐奎刘莎莎
Owner 中农种源(深圳)科技有限公司
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