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Production method and applications of autosomal dominant polycystic kidney disease gene mutation pig

A production method and autosomal technology, applied to the production method and application field of autosomal dominant polycystic kidney disease gene mutation pigs, can solve the problems of low targeting efficiency, not very wide application of gene targeting technology, lack of embryonic stem cells, etc.

Active Publication Date: 2015-07-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although gene targeting has many advantages, due to the low targeting efficiency (10 -6 ) and the lack of embryonic stem cells, the application of gene targeting technology in other species is not very extensive

Method used

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  • Production method and applications of autosomal dominant polycystic kidney disease gene mutation pig
  • Production method and applications of autosomal dominant polycystic kidney disease gene mutation pig
  • Production method and applications of autosomal dominant polycystic kidney disease gene mutation pig

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Cloning of porcine polycystic kidney disease gene and screening of mutation sites

[0031] Based on the mRNA sequence of the porcine polycystic kidney disease gene PKD1 (GenBank Accession No.: NM_001246202), the inventor used ZFN, Talen and CRISPR-Cas9 gene editing techniques to locate the 642-643 site of exon 5 of PKD1 Introducing insertion mutations ( figure 1 ). As a target site for gene editing, the resulting mutant protein would fail to retain most of its functional domains, thus rendering PKD1 non-functional.

Embodiment 2

[0032] Example 2 Screening of PKD1 knockout fibroblasts

[0033] According to the target sites determined in Example 1, ZFNs for gene editing can be designed. Talen and CRISPR-Cas9 technologies can also be used for gene editing operations. ZFN technology is preferred in this embodiment. After obtaining ZFN, it is first necessary to optimize the amount of fibroblast transfected. We used 0.3 μg, 0.5 μg, 1 μg, 2 μg, 4 μg of ZFN plasmids (ZFN transfection is a pair of plasmids, and the amount at this time represents the amount of each plasmid) to transfect 1×10 6a fibroblast. The culture condition of fibroblasts was 37°C, the medium was DMEM (Sigma) plus 10% fetal bovine serum (Gibco), and the medium was changed 24 hours before transfection, so that the cells were in a state of exponential growth. Good transfection conditions are based on the following two criteria: 1) The fibroblasts are in good condition after transfection, with normal morphology and no large number of deaths;...

Embodiment 3

[0037] Example 3 Preparation of PKD1 Gene Knockout Minipigs by Somatic Cell Nuclear Transfer Technology

[0038] The nuclei of the screened positive clones were transferred into the enucleated oocytes by somatic cell nuclear transfer technology, cultured in vitro to form reconstituted embryos, and then transplanted into each recipient pig with an average of 384 reconstituted embryos In the oviducts of 13 recipient pigs. After about 114 days, 5 surrogate sows gave birth to 20 cloned miniature pigs ( image 3 ).

[0039] The operation steps are as follows: Retrieve the pig ovary from the slaughterhouse → extract the follicles with a needle tube → put the follicles into the maturation solution for in vitro maturation → after the fibroblasts at the mutant clone point fill the culture wells, digest, wash and resuspend → recipient oocytes Denucleate under the microscope, then inject the donor cells into the perivitelline space so that the cell membranes of the two are in close con...

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Abstract

The present invention provides a production method and applications of an autosomal dominant polycystic kidney disease gene mutation pig, wherein a gene editing technology is used to introduce insertion mutation into the 5# exon of the pig PKD1 gene and knockout the pig PKD1 gene to construct an autosomal dominant polycystic kidney disease gene mutation pig model. The constructed gene mutation pig model of the present invention can be used for research and drug screening of the autosomal dominant polycystic kidney disease.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a production method and application of autosomal dominant polycystic kidney disease gene mutant pigs. Background technique [0002] Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary polycystic kidney disease with an incidence rate of 1 / 400-1 / 1000 in the population. ADPKD is characterized by fluid-filled vesicles in bilateral kidneys, accompanied by a variety of extrarenal symptoms. As the disease progresses, renal parenchyma is gradually replaced by vesicles and fibrotic tissue, leading to loss of renal function. The clinical manifestations of patients usually appear in the age of 20 to 30, and only less than 2% of the cases appear in the fetus or childhood. At the age of 60, about 50% of patients will develop end-stage renal disease (ESRD), which accounts for 5% of all ESRD cases, which is also the fourth leading cause of end-stage renal disease in my coun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/873
Inventor 李宁贺津胡晓湘赵要风李秋艳于政权
Owner CHINA AGRI UNIV
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