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A kind of preparation method of rosa26 site-directed knock-in human complement regulatory protein minipig

A technology that regulates proteins, px330-prosa26-grna, is applied in the field of genetic engineering, which can solve problems such as immune rejection, and achieve the effect of promoting the research and development process and avoiding unclear genetic background

Active Publication Date: 2022-05-06
INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although pigs are excellent donors for xenotransplantation, immune rejection will occur in xenotransplantation from pigs to primates, which is also one of the important problems to be solved in xenotransplantation research

Method used

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  • A kind of preparation method of rosa26 site-directed knock-in human complement regulatory protein minipig
  • A kind of preparation method of rosa26 site-directed knock-in human complement regulatory protein minipig
  • A kind of preparation method of rosa26 site-directed knock-in human complement regulatory protein minipig

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1 Porcine Rosa26 site CRISPR / Cas9 gRNA screening and homologous recombination donor vector construction

[0101] 1. Construction of pig Rosa26 site CRISPR / Cas9 expression vector and gRNA screening

[0102] The porcine Rosa26 locus is located on chromosome 13, and the genome sequence information of Rosa26 was obtained according to the genome sequence information in the Ensemble and UCSC databases. According to the mechanism of action of CRISPR / Cas9, a total of 6 gRNA candidate sites targeting porcine Rosa26 intron 1 were designed through the online design website, and the sequences of the 6 guide RNAs (Guide RNA, gRNA) are shown in Table 1.

[0103] Table 1 Candidate site sequences of pRosa26 gRNA

[0104]

[0105] Select px330 as the backbone vector, and the px330 is a pX330 vector capable of carrying CRISPR / Cas9, px330 is digested with the restriction endonuclease BbsI, and bathed in water at 37°C for 4 h. The digested products were subjected to agarose ge...

Embodiment 2

[0112] Example 2 Transfection of pig fetal fibroblasts and screening of monoclonal target cells

[0113] 1. Establishment of porcine fetal fibroblast lines

[0114] The 35-day pregnant Bama miniature pig was anesthetized, and the fetus was aseptically removed from the uterus. After washing the fetus with PBS containing double antibodies, it was placed in an ultra-clean workbench, and the head, limbs, internal organs and organs of the fetus were removed with ophthalmic scissors. Wash the cartilage tissue with PBS; add an appropriate amount of FBS to the cell culture dish to keep the tissue from being too dry, and use ophthalmic scissors to cut the remaining tissue as much as possible. Add 8-10 mL of collagenase solution to each fetal sample, digest at 37°C for 4 h, take it out and shake it several times during the period.

[0115] Add the digested tissue pieces to 10 mL DMEM containing 15% FBS to stop the digestion, centrifuge at 1000 rpm for 5 min, discard the supernatant, an...

Embodiment 3

[0120] Example 3 Identification of Single Clones of Site-directed Knock-in hCRP Positive Cells

[0121] Due to the double-strand break caused by Cas9 cleavage, under the mediation of homologous recombination vector, the HDR repair method will accurately insert the genome, so it is necessary to use the genome of the single clone of the target cell as a template to perform PCR amplification of the exogenous gene respectively , and sequence the 5' and 3' junction regions of homologous recombination to detect gene editing.

[0122] The px330-Rosa26-4gRNA (4 µg) and the corresponding homologous recombination targeting pGSI-Rosa26-hCRP (6 µg) vector DNA were electrotransfected into pig fetal fibroblasts. Monoclonal cell lines were selected after 10-15 days by diluting the cells and adding puromycin-resistant drugs. The genotypes of cells obtained from each monoclonal source were detected, and the primers for PCR amplification are shown in Table 3. Genomic DNA of 27, 20 and 21 mono...

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Abstract

The invention discloses a method for preparing a human complement regulatory protein miniature pig at the Rosa26 site. The invention inserts the human complement regulatory protein gene into the pig ROSA26 gene site through site-directed gene recombination, thereby constructing The minipigs constitutively expressing human complement regulatory proteins were obtained. The constructed minipigs not only avoided the unclear genetic background caused by the random insertion of foreign sources and the unstable expression level caused by the segregation of chromosomes during the breeding process of offspring, but also laid a solid foundation for future development. The study of xenotransplantation and immune-related diseases provides important animal resources and has important clinical application value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular, the invention relates to a preparation method for knocking in human complement regulatory protein minipigs at the Rosa26 site. Background technique [0002] For patients with organ failure, life can be saved through artificial mechanical devices, stem cell organ regeneration or organ transplantation. However, the precision of artificial mechanical devices is not high, and it cannot match the excellent performance of organs. The research on embryonic stem cells induced to develop into organs has not yet achieved breakthroughs. Therefore, organ transplantation is still an excellent means of treating patients with organ failure. With the development of medical technology, organ transplantation has become an effective method for the treatment of end-stage organ failure and has been widely used clinically. However, the shortage of donor organs has gradually become prominent....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/12C12N15/85C12N15/65C12N15/55C12N15/877C12N15/90A01K67/027
CPCC12N15/113C12N15/8509C12N15/907C12N15/65C12N9/22C12N15/8778A01K67/0276A01K67/0278C12N2310/20A01K2207/15A01K2217/072A01K2217/075A01K2227/108A01K2267/025A01K2267/0318
Inventor 秦川卢天宇邓守龙杨博超于佳楠王若琳徐亚新
Owner INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI
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