Method for knocking out bovine myostatin gene by using zinc finger nuclease

A myostatin and zinc finger nuclease technology, applied in the field of genetic engineering, can solve the problems of inability to obtain positive single-cell clones, long cycle and high risk, simplify the biosafety evaluation process, save the drug screening process, and avoid confrontation. The effects of drug poisoning

Inactive Publication Date: 2013-09-18
BEIJING GEFUCURE BIOTECHNOLOGY LIMITED COMPANY
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

And in the process of cell screening, some can't even get positive single cell clones
If three clonings are required to achieve gene knockout cattle without resistance genes, it will take at least 42 months, which is a long cycle and high risk

Method used

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  • Method for knocking out bovine myostatin gene by using zinc finger nuclease
  • Method for knocking out bovine myostatin gene by using zinc finger nuclease
  • Method for knocking out bovine myostatin gene by using zinc finger nuclease

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Effect test

Embodiment 1Z

[0031] Screening and knockout efficiency of embodiment 1 ZFN expression vector

[0032] 1. ZFN expression vector construction

[0033] MSTN (NC 007300.4) gene sequence information was obtained from the NCBI website, ZFNs design was completed by Sigma Company, and the designed ZFNs sites were located on exons 1 and 3. MSTN-ZFNs-Set 1 acts on the first exon, and MSTN-ZFNs-Set 2 and MSTN-ZFNs-Set 3 act on the third exon. The DNA sequences they act on are:

[0034] ZFNs-Set1: GTCATTACCATGCCCACGG AGTGTG AGTAGTCCTGCTGGT ;

[0035] ZFNs-Set 2: CTCATCAAACCCATGA AAGACGG TACAAGGTATACTGG ;

[0036] ZFNs-Set 3: TTCCCAGAAC CAGGA GAAGATGGACTGGTA

[0037] The underlined parts are zinc finger protein binding sequences, and the middle part is the FokI endonuclease cleavage site. The corresponding three ZFNs expression vectors are: MSTN-ZFN-Set1-pZFN1 / pZFN2, MSTN-ZFN-Set2-pZFN1 / pZFN2 and MSTN-ZFN-Set3-pZFN1 / pZFN2.

[0038] ZFN expression vector construction process: Taking MST...

Embodiment 2

[0045] Example 2 Obtaining of Single Cell Clones and Identification of Gene Knockout Clones

[0046] 1. Obtaining of single cell clones

[0047] Using the AMAXA electroporation instrument to electrotransfer bovine fibroblasts, using optimized electrotransfer parameters T-016, the gene transfection efficiency can reach more than 90%. The transfected genetic material is mRNA, and the half-life in the cell is about 8 hours. There will be no random insertion into the cell genome when the DNA is transfected, which has a good guarantee for the genetic stability of the animal. At the same time, there will be no random integration of resistance genes, which meets the requirements of biological safety.

[0048] Specific operation method: Recover and purify the in vitro transcribed mRNA with a kit from Applied Biosystems, elute and dissolve with DEPC water, and make the final concentration about 500 ng / μl. The mRNA corresponding to a pair of ZFNs is 2 μg each, the total mRNA amount is...

Embodiment 3

[0052] Example 3 Preparation of embryos of gene knockout single-cell clones and cloned cattle

[0053] 1. Preparation of knockout cattle

[0054] The specific process includes:

[0055] (1) Holstein cow fetal fibroblast culture

[0056] Fetal ear tissues of 40-day-old Holstein cows were taken, and the bovine fetal fibroblast cell line was established through primary culture, subculture, freezing and other in vitro culture operations.

[0057] (2) Gene knockout single cell clone

[0058] The acquisition of gene knockout single cell clones is the same as in Example 2.

[0059] (3) Transgenic cloned embryo preparation and embryo transfer

[0060]Collect the ovaries of adult cattle from the slaughterhouse, take follicles with a diameter of 2-8 mm, and recover the cumulus-oocyte-complex with uniform shape and compact structure, and divide the cumulus-oocyte-complex into 50-60 pieces Put each hole into a four-well plate containing maturation solution (M199+10% fetal bovine seru...

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Abstract

The invention provides a method for knocking out a bovine myostatin gene by using zinc finger nuclease. The method comprises the following steps of: designing a ZFNs specific site expression vector according to a bovine myostatin gene sequence and transferring into bovine fibroblasts to obtain cells in which the myostatin gene is knocked out. The one-time transfection can be realized by utilizingZFNs-mediated gene knockout so as to obtain cell clone in which a diallele is knocked out, which is difficult to realize in the conventional gene targeting process; therefore, the medicine screening process is saved, the formation of cell monoclone is facilitated, the process that cells are required to resist the poisoning of medicines is avoided, key effects are achieved on the subsequent somatic cell nuclear transplantation and the development quality of embryos, resistance genes are not contained at the same time, and the biological safety evaluation process is greatly simplified.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for knocking out bovine myostatin gene by using zinc finger nuclease. Background technique [0002] Gene targeting technology is a technology that changes the genetic information of organisms in a targeted manner. The two limiting factors of conventional gene targeting animal production are: first, the gene targeting efficiency of animal somatic cells is very low, 10 -6 ~10 -7 ; Second, the time period for producing biallelic knockout animals is long and the cloning efficiency is low. With the continuous development of molecular biology, many new technologies and methods have emerged to improve the efficiency of gene targeting, such as the use of gene targeting strategies without promoter selection, the isolation and induction of stem cells in animals, and so on. However, these improvements have not significantly promoted the production of gene-targeted ani...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/877A01K67/027
Inventor 于胜利罗俊杰丁方荣李松汤波李宁
Owner BEIJING GEFUCURE BIOTECHNOLOGY LIMITED COMPANY
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