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Method for knocking out selective marker gene of transgenic pig

A technology for screening marker genes and transgenic pigs, applied in the field of genetic engineering

Active Publication Date: 2015-03-18
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no related research on marker free pigs. Due to its importance, the research on transgenic pigs with marker gene knockout is particularly important

Method used

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  • Method for knocking out selective marker gene of transgenic pig
  • Method for knocking out selective marker gene of transgenic pig
  • Method for knocking out selective marker gene of transgenic pig

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of self-shearing element PCN

[0029] The pig sperm-specific promoter was constructed into the pUC57 cloning vector (provided by the University of Utah, USA) for gene synthesis, named pGENE1 vector (this step was performed by the University of Utah, USA), and double-digested with SalI+AgeI enzymes, the system was 50ul: Carrier 2ul, enzyme 2ul each, 10xbuffer4 plus 5ul, sterile water 31ul, enzyme digestion at 37°C for 2 hours, gel run recovery to obtain a 931bp target fragment, sequenced (nucleotide sequence shown in SEQ ID No.1); and then pACN The carrier (provided by the University of Utah, USA) was double-digested with SalI+AgeI enzyme, and the system was 50ul: 2ul of vector, 2ul of each enzyme, 5ul of 10xbuffer4, 31ul of sterile water, digested at 37°C for 2h, and recovered the target fragment of 5805bp by running the gel ; Ligate the two fragments recovered above, 10ul of the system: 6ul of the recovered small fragment, 2ul of the large fragme...

Embodiment 2

[0030] Example 2 MSTN: construction of marker free targeting vector

[0031] 2. Double-cut the correctly constructed pPCN vector with NheI+NotI, and the system is 50ul: 2ul of vector, 2ul of each enzyme, 5ul of 10xbuffer4, 31ul of sterile water, enzyme digestion at 37°C for 2h, running gel to recover 3620bp Fragment; at the same time, the original pMSTN targeting vector in the laboratory is 50ul with NheI+NotI double enzyme digestion system: 2ul of vector, 2ul of each enzyme, 5ul of 10xbuffer4, 31ul of sterile water, enzyme digestion at 37°C for 2h, running the gel to recover the target 14580 fragments to obtain Backbone fragment, system 10ul: recovered small fragment 6ul, large fragment 2ul, 10xbuffer plus 1ul, T4 ligase 1ul, ligated overnight at 16. The final marker free targeting vector pMSTN-PCN1 vector (nucleotide sequence shown in SEQ ID No.3) for transformation, shaking, small body plasmid, enzyme and positive identification is used for the next step of the experiment (...

Embodiment 3

[0031] 2. Double-cut the correctly constructed pPCN vector with NheI+NotI, and the system is 50ul: 2ul of vector, 2ul of each enzyme, 5ul of 10xbuffer4, 31ul of sterile water, enzyme digestion at 37°C for 2h, running gel to recover 3620bp Fragment; at the same time, the original pMSTN targeting vector in the laboratory is 50ul with NheI+NotI double enzyme digestion system: 2ul of vector, 2ul of each enzyme, 5ul of 10xbuffer4, 31ul of sterile water, enzyme digestion at 37°C for 2h, running the gel to recover the target 14580 fragments to obtain Backbone fragment, system 10ul: recovered small fragment 6ul, large fragment 2ul, 10xbuffer plus 1ul, T4 ligase 1ul, ligated overnight at 16. The final marker free targeting vector pMSTN-PCN1 vector (nucleotide sequence shown in SEQ ID No.3) for transformation, shaking, small body plasmid, enzyme and positive identification is used for the next step of the experiment (see the vector structure for details) figure 2 ). Example 3 Acquisit...

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Abstract

The invention belongs to the field of genetic engineering, and provides a method for knocking out a selective marker gene of a transgenic pig; the method disclosed by the invention comprises the following steps: promoting a Cre / loxp site specific recombinase system by utilizing a pig sperm specific included promoter, establishing a self-shearing component PCN (Personal Communication Network), normally carrying out targeting and screening positive clone, after carrying out SCNT (Somatic Cell Nuclear Transfer) to obtain founder positive transgenic pig, and self-shearing to knock out the marker gene through hybridization, wherein in the self-shearing component PCN, P represents the pig sperm specific promoter; C represents a Cre enzyme gene; and N represents a screened marker neo gene required while targeting. Because Cre enzyme is specifically promoted after sperm is mature, the pig, the selective marker neo gene of which is knocked out, can be obtained; and thus, the pig, the selective marker gene of which is knocked out, can be conveniently obtained.

Description

technical field [0001] The invention relates to genetic engineering, in particular to a method for knocking out a genetically modified pig screening marker gene. Background technique [0002] In the process of biological development, there have been important revolutionary breakthroughs in many aspects. For example: mouse gene targeting technology, the successful separation of mouse and human embryonic stem cells (ES cells), the completion of the human genome project and the mouse genome project, more than 30,000 human genes are considered to be the "book" that determines the fate of human beings, displayed in front of humans. Therefore, the functional gene research using the mouse model lays an important foundation for understanding the human gene function, but due to the limitations of the mouse model, it cannot meet the current research. However, large animals, especially pigs, are currently considered ideal animal models due to their physiological characteristics simil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A61D19/00A01K67/027C12Q1/68
Inventor 李宁孙照霖康倩倩吴森李秋艳温啸赵蕊
Owner CHINA AGRI UNIV
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