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Swine pseudo attp site and use of swine pseudo attp site

A promoter and optional technology, applied in the field of bioengineering, can solve problems such as unfavorable cultivation of stable strains, inactivation, destruction of endogenous genes, etc.

Active Publication Date: 2012-07-04
深圳华大基因农业控股有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Most of the existing animal transgenic technologies are based on random integration. On the one hand, the integration efficiency is low, and the site is unpredictable; Random insertion in the host genome may lead to the destruction or inactivation of endogenous genes, and may also activate some genes that are normally closed, easily causing animal abnormalities
These have greatly affected the application of transgenic technology
[0003] The current animal transgenic technology still needs to be improved

Method used

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  • Swine pseudo attp site and use of swine pseudo attp site
  • Swine pseudo attp site and use of swine pseudo attp site
  • Swine pseudo attp site and use of swine pseudo attp site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, the construction of the pCMV-Int vector containing streptomyces phage ΦC31 integrase gene

[0064] (1) Synthesis of Streptomyces phage φC31 integrase gene (Int gene): Submit the sequence shown in SEQ ID NO: 5 to Shanghai Jierui Biotechnology Co., Ltd. for Int gene synthesis, and construct the synthesized Int gene in pGH vector On, get the pGH-Int plasmid.

[0065] (2) Enzyme digestion of pGH-Int plasmid: Take 20 microliters (400-500ng / ul) of pGH-Int plasmid for enzyme digestion. The enzyme digestion system is:

[0066] pGH-Int plasmid

20 microliters

10xNEB buffer2

5 microliters

endonuclease NheI

1 microliter

endonuclease XhoI

1 microliter

[0067] 100xBSA

0.5 μl

double distilled water

22.5 µl

total reaction volume

50 µl.

[0068] Place the above enzyme digestion system at 37°C and digest overnight.

[0069] (3) Enzyme digestion of pcDNA3.1 plasmid: take 10 mic...

Embodiment 2

[0076] Embodiment 2, the construction of pattB-CAG-Intron-EGFP carrier

[0077](1) Synthesis of attB and Intron gene fragments: Submit the sequence Intron (intron) sequence and the attB sequence shown in SEQ ID NO: 4 to Shanghai Jierui Biotechnology Co., Ltd. for gene synthesis, and construct the synthesized genes separately On the pGH vector, pGH-attB and pGH-Intron plasmids were obtained, respectively. Among them, the NCBI of the used intron is AF369966.1, and the full length of the sequence is

[0078] CGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGC...

Embodiment 3

[0084] Embodiment 3, cell transfection and screening

[0085] The porcine kidney epithelial cell line PK15 was inoculated into a 6-well plate at a density of 30%, and the cells were cultured until the confluence was 70-80%, and transfection was started.

[0086] (1) Transfection: The pCMV-Int plasmid obtained in Example 1 and the pattB-CAG-Intron-EGFP plasmid obtained in Example 2 were mixed in different ratios, and the liposome LF2000 kit was used to follow the manufacturer's instructions. Instructions for transfecting PK15 cells.

[0087] (2) Exploration of the effect of two plasmid ratios: with the quality of the pattB-CAG-Intron-EGFP plasmid constant at 1 microgram, by selecting the quality of the pCMV-Int plasmid as 1 microgram, 2 micrograms, 3 micrograms, 4 micrograms, and 5 micrograms, The pattB-CAG-Intron-EGFP: pCMV-Int was transfected into PK15 cells according to different ratios of 1:1, 1:2, 1:3, 1:4, and 1:5. The best mixing ratio, the result is that the fluoresce...

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Abstract

The invention relates to a swine pseudo attp site and use of the swine pseudo attp site. More specifically, the invention relates to a separate oligonucleotide capable of being recognized by a phiC31 integrase, a vector suitable for transforming a swine cell, a group of vectors suitable for transforming the swine cell, a transgenic swine cell or a culture of the transgenic swine cell, a method for preparing the transgenic swine cell and a sequence for determining the pseudo attp site in a swine genome, wherein the separate oligonucleotide capable of being recognized by the phiC31 integrase contains a nucleotide sequence shown as SEQ ID NO:3. According to the invention, exogenous genes can be effectively imported into the swine cell under the mediation of the phiC31 integrase.

Description

technical field [0001] The invention relates to the technical field of bioengineering. In particular, the invention relates to porcine pseudo attp sites and uses thereof. More specifically, the present invention relates to an isolated oligonucleotide that can be recognized by ΦC31 integrase, a vector suitable for transforming pig cells, a group of vectors suitable for transforming pig cells, a transgenic pig cell or its cultures, a method for producing transgenic porcine cells, and a method for determining the sequence of pseudo attp sites in the porcine genome. Background technique [0002] Most of the existing animal transgenic technologies are based on random integration. On the one hand, the integration efficiency is low, and the site is unpredictable; Random insertion in the host genome may lead to the destruction or inactivation of endogenous genes, and may also activate some genes that are normally closed, easily causing animal abnormalities. These have greatly aff...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/85C12N5/10C12Q1/68
Inventor 李勇刘欢张欢欢张楚新杜玉涛王俊汪建杨焕明
Owner 深圳华大基因农业控股有限公司
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