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Targeting vector for pig GHR gene knock-out and applications thereof

A technology for targeting vectors and genes, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as huge differences in bones.

Inactive Publication Date: 2018-12-07
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the differences between mice and humans, there is a huge difference between mice and humans in the study of bone growth and fat metabolism

Method used

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  • Targeting vector for pig GHR gene knock-out and applications thereof
  • Targeting vector for pig GHR gene knock-out and applications thereof
  • Targeting vector for pig GHR gene knock-out and applications thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] In this example, CRISPR / Cas9 technology was used to prepare porcine GHR gene knockout cells, and the specific experimental steps were as follows:

[0035] 1. Expression vector construction

[0036] The backbone vector used is the pX330 vector, U1-U3 sgRNA is designed for part of the 10th exon, and D1-D3 sgRNA is designed for the 3′UTR. In addition, in order to distinguish the dwarf phenotype caused by the deletion of part of the 10th exon or the deletion of the 3′UTR region, Design the M1-M4 sgRNA after the first polyA of exon 10, and the specific construction process of the sgRNA expression vector is as follows:

[0037] The two oligonucleotide fragments of each target sequence in Table 1 were diluted to 200 mM, mixed, annealed to form a double strand, connected to the pX330 backbone carrier obtained after digestion with BbsI enzyme (NEB (USA) company), and Sequencing verification obtained 10 recombinant vectors with correct sequences.

[0038] Table 1 sgRNA sequence...

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PUM

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Abstract

The invention belongs to the technical field of biology, and specifically discloses a targeting vector for pig GHR gene knock-out and applications thereof. The targeting vector is a sgRNA expression vector based on a CRISPR / Cas system; wherein the target sequences of sgRNA are arranged on the 10th exon and 3'UTR of a pig GHR gene. The provided targeting vector is applied to targeting knock-out ofpig GHR gene, and the knock-out efficiency can reach 12.7%. After the GHR gene is successfully knocked out, somatic cells can be used to establish a GHR gene knock-out pig model by a somatic cell nuclear transplanting technology, and materials are provided for the research of physiological action and molecular mechanism of GHR gene.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a targeting carrier for knocking out porcine GHR gene and a method for preparing a porcine cell line for knocking out GHR gene. Background technique [0002] Laron syndrome (Laron syndrome) is a rare autosomal recessive genetic disease caused by growth hormone receptor (GHR) mutations. This mutation causes the obstacle of growth hormone signal transduction through GHR, which in turn affects the production of insulin-like growth factor 1 (IGF1), and ultimately affects the growth-promoting, metabolic and developmental effects of growth hormone on the body. The main symptoms of the disease are (1) dwarfism; (2) low levels of growth hormone and high levels of IGF1 in the body. At present, the disease is mainly treated clinically by supplementing IGF1 to patients, but long-term supplementation of IGF1 will inevitably produce a series of side effects, such as obesity, hypoglyc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N9/22C12N15/877A01K67/027
CPCA01K67/0275A01K2207/15A01K2227/108A01K2267/0306C12N9/22C12N15/8778C12N15/907
Inventor 张然韩琪陈慧玲
Owner CHINA AGRI UNIV
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