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Method for knocking out cattle beta-lactoglobulin gene by using zinc finger nucleases (ZFNs)

A technology of zinc finger nuclease and lactoglobulin, applied in the field of genetic engineering, can solve the problems of inability to obtain positive single cell clones, high risk, long cycle, etc., and achieve the effect of simplifying the biosafety evaluation process

Inactive Publication Date: 2011-10-12
BEIJING GEFUCURE BIOTECHNOLOGY LIMITED COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • 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 cattle beta-lactoglobulin gene by using zinc finger nucleases (ZFNs)
  • Method for knocking out cattle beta-lactoglobulin gene by using zinc finger nucleases (ZFNs)
  • Method for knocking out cattle beta-lactoglobulin gene by using zinc finger nucleases (ZFNs)

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Experimental program
Comparison scheme
Effect test

Embodiment 1Z

[0034] Screening and knockout efficiency of embodiment 1 ZFN expression vector

[0035] 1. Screening of ZFNs

[0036] BLG (NC_007309.4) gene sequence information was obtained from the NCBI website, and the ZFNs design was completed by Sigma Company, and the designed ZFNs sites were located on exons 1 and 2. ZFNs-Set1 and ZFNs-Set 2 act on the first exon, and ZFNs-Set 3 acts on the second exon. The DNA sequences they act on are:

[0037] ZFNs-Set 1: CC CAGGCCCTCATTGTC ACCC AGACCATGAAGGGCCTG GA;

[0038] ZFNs-Set 2: AG GCCCTCATTGTCAC CCAGACC ATGAAGGGCCTGGAT AT;

[0039] ZFNs-Set 3: CC CAGAGTGCCCCCCTGAGA GTGTA TGTGGAGGAGCTGAA GC.

[0040] 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: PZFN1 / PZFN2-set 1, PZFN1 / PZFN2-set 2 and PZFN1 / PZFN2-set 3. All three expression vectors can function in yeast, refer to Doyon et al., Nat Biotechnol, (...

Embodiment 2

[0053] Example 2 Obtaining of Single Cell Clones and Identification of Gene Knockout Clones

[0054] 1. Obtaining of single cell clones

[0055] 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.

[0056] Specific operation method: using the plasmid pBudCE-ZFN1-2 as a template, the in vitro transcribed mRNA was recovered and purified with a kit from Applied Biosystems, and eluted with DEPC water to make the final concentration about 500 ng / μl. The mRNA corresponding to a pair o...

Embodiment 3

[0060] Example 3 Preparation of embryos of gene knockout single-cell clones and cloned cattle

[0061] 1. Preparation of knockout cattle

[0062] The specific process includes:

[0063] (1) Holstein cow fetal fibroblast culture

[0064] 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.

[0065] (2) Gene knockout single cell clone

[0066] The acquisition of gene knockout single cell clones is the same as in Example 2.

[0067] (3) Transgenic cloned embryo preparation and embryo transfer

[0068] 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 ser...

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Abstract

The invention provides a method for knocking out a cattle beta-lactoglobulin gene by using zinc finger nucleases (ZFNs), comprising the following steps of: according to a cattle beta-lactoglobulin gene sequence, designing ZFNs specific site expression vector and transplanting the ZFNs specific site expression vector into cattle fibroblast; and obtaining cells with the beta-lactoglobulin gene knocked out. By using ZFNs mediated gene knockout, one-time transfection can be realized so as to obtain cell clones with biallelic genes knocked out, which is difficultly achieved in the conventional gene targeting process. The drug screening process is saved. The method disclosed by the invention is advantageous for forming monoclonal cells. The process required by cells for resisting drug toxic process is avoided. The method plays a key role in the improvement of subsequent somatic cell nuclear transplantation efficiency and embryonic development quality. Simultaneously, resistance genes are not contained; 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 β-lactoglobulin 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-target...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N5/071C12N15/877A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/101C07K14/4717C12N9/22C12N15/8509
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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