Bovine genome pseudo-attP site and its use

A genome and gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., to achieve the effect of promoting further application

Active Publication Date: 2009-08-12
SHANGHAI JIAO TONG UNIV AFFILIATED CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether there is a false attP site recognized by phage φC31 integrase in the bovine genome has not been reported yet

Method used

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  • Bovine genome pseudo-attP site and its use
  • Bovine genome pseudo-attP site and its use
  • Bovine genome pseudo-attP site and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 , Construction of plasmid pCMVInt containing Streptomyces phage φC31 integrase gene

[0028] According to the method described in the literature (Groth A.C., Olivares E.C., Thyagarajan B.and Calos M.P..Proc.Natl Acad.Sci.USA 97(2000):5995-6000), construct the plasmid pCMVInt containing the integrase gene of Streptomyces phage φC31 , the construction is divided into two steps, as follows:

[0029] The first step, the construction of plasmid pTA-Int:

[0030](1) Genomic DNA of Streptomyces phage φC31 (German Collection Center for Microbiology and Cell Culture, Braunschweig, Germany, DSM No. 49156) was used as a template to amplify the integrase gene by PCR.

[0031] The primer sequences are:

[0032] 5'-GAGTTCTCTCAGTACTAGTCGTAGGGTCGC-3'; and

[0033] 5'-CGCCTAACAGGGGATCCGGTGTCTCGCTAC-3';

[0034] PCR reaction system: 1×PCR buffer solution (TaKaRa company), 1.5mM Mg 2+ , 10 μM each primer, 200 μM dNTPs, 100-200 ng template DNA, 2.5 U LATaq DNA polymerase (...

Embodiment 2

[0047] Example 2 , construction of plasmid vector containing attB site

[0048] According to the method described in the literature (Groth A.C., Olivares E.C., Thyagarajan B.and Calos M.P..Proc.Natl Acad.Sci.USA 97(2000):5995-6000), construct the plasmid pBCPB+ containing the attB site, the specific process is as follows :

[0049] The first step, the construction of plasmid pTA-attB:

[0050] (1) With a pair of primers:

[0051] 5'-CAGGTACCGTCGACGATGTAGGTCACGGTC-3'; and

[0052] 5'-GTCGACATGCCCGCCGTGACCG-3'

[0053] Using the genomic DNA of Streptomyces lividans (German Microbiology and Cell Culture Collection Center, Braunschweig, Germany, DSM No.46482) as a template, a 293bp sequence was amplified by PCR, which contained the φC31 integrase recognition attB site.

[0054] PCR reaction system: 1×PCR buffer solution (TaKaRa company), 1.5mM Mg 2+ , 10 μM each primer, 200 μM dNTPs, 100-400 ng template DNA, 2U Taq DNA polymerase (TaKaRa Company).

[0055] PCR reaction co...

Embodiment 3

[0099] Example 3 , construction of a plasmid containing the gene of interest (EGFP) and attB site

[0100] In this example, unless otherwise specified, all restriction endonucleases were purchased from TaKaRa Company.

[0101] The construction of plasmid pEGFP-N1-attB is divided into three steps:

[0102] Step 1: Construction of plasmid pSL301-attB

[0103] (1) Restriction plasmid pBCPB: pBCPB obtained in Example 2, 25 μl; Xho I, 4 μl; 10×H, 20 μl; sterilized double distilled water, 151 μl; total reaction volume, 200 μl. 37°C overnight.

[0104] (2) Restriction plasmid pSL301: pSL301 (Invitrogen), 25 μl; Xho I, 4 μl; 10×H, 20 μl; sterilized double distilled water, 151 μl; total reaction volume 200 μl. 37°C overnight.

[0105] (3) Dephosphorylation reaction of the linearized fragment of plasmid pSL301: Precipitate the digested product of pSL301 with 2 times the volume of absolute ethanol, wash once with 70% ethanol, dissolve the linearized vector in 43 μl sterile double d...

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Abstract

Method for separating and determining bovine genome pseudo attp site and its usage are disclosed. The process is carried out by separating out tow pseudo attp sites from bovine gene, having 38% and 41% homology with attp site of streptomycete phagicin phiC31 genome, discriminating by streptomycete phagicin phiC31 integrase and integrating foreign gene containing attB site with the site of bovine geneome under mediation of integrase. It has higher integration efficiency.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a false attP site recognizable by Streptomyces phage φC31 integrase in bovine genome and its application for integrating exogenous genes in bovine cells. Background technique [0002] Transgenic technology has a wide range of applications in the field of bioengineering. In addition to applying a variety of physical (such as electric shock) and chemical (such as cationic liposome) methods to introduce foreign genes into the cell genome, people also use a variety of tool enzymes to achieve gene transfer. For example, the Cre recombinase of coliphage P1 works efficiently in yeast, mammalian and plant cells, mouse embryonic stem cells and mice, and recombinases such as FLP and β can also efficiently perform homologous recombination at specific sites. [0003] Recently, Streptomyces phage phiC31 integrase (phiC31 integrase) has become a powerful tool in genetic modification ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/12C12N15/09C12N15/85
Inventor 曾溢滔黄淑帧马晴雯
Owner SHANGHAI JIAO TONG UNIV AFFILIATED CHILDRENS HOSPITAL
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