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Gene target vector based on bird gland related virus and construction method thereof

A technology of gene targeting and construction method, which is applied in the direction of microorganism-based methods, genetic engineering, plant gene improvement, etc., can solve problems such as targeting failure, achieve the effects of inhibiting random integration, improving efficiency, and improving the efficiency of homologous recombination of positive clones

Inactive Publication Date: 2009-08-12
JINLING INST OF TECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the promoter of the ovalbumin gene (OV) with the highest expression level in poultry eggs is an important candidate promoter for production of oviduct bioreactors, but for gene targeting of OV, the position effect may be the most important factor leading to the failure of targeting

Method used

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  • Gene target vector based on bird gland related virus and construction method thereof
  • Gene target vector based on bird gland related virus and construction method thereof
  • Gene target vector based on bird gland related virus and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Construction of AAAV gene transfer vector (pAAAV-TR).

[0030] The basic skeleton of the AAAV transfer vector is obtained by PCR amplification of the pTRE-tight-B1 plasmid (purchased from Clontech Company), and the primer sequences are respectively:

[0031] Primer 1: aa aagctt aaggatccatatgcggccg ctcgag aac agatct aGAGCTTCCTCGCTCACTGACTCG, the lowercase letters are the introduced multiple cloning sites, and the underlines indicate the HindIII, XhoI, and BglII sites in turn.

[0032] Primer 2: ttt gaattc The lowercase letters of tagACGTCAGGTGGCACTTTTC are the restriction restriction sites introduced, and the underline indicates the EcoR I site.

[0033] 20 μL reaction system for PCR includes: 10 ng plasmid DNA, 2 μl 10×LA PCR buffer, 0.4 mM dNTPs, 10 μM primers, 2 units of TaKaRa LA Taq DNA polymerase (purchased from TaKaRa Company). The PCR reaction conditions were: denaturation at 94°C for 2 minutes; 32 cycles of 94°C for 30s, 56°C for 30s, and 72°...

Embodiment 2

[0035] Example 2: Amplification and doubling of HS4.

[0036] Primers were designed according to the chicken HS4 sequence (GenBank NW_001471556 293787-294082):

[0037] Primer 3: CCGCGGG TGATCA CGGGGAGAG is underlined to indicate the Bcl I site

[0038] Primer 4: TTCAGCCT AGATCT TTTTCCCCGTA is underlined to indicate the BglII site

[0039] PCR is a 20 μL reaction system including: 50 ng chicken genomic DNA, 10 μl 2×GC buffer, 0.4 mM dNTPs, 10 μM primers, and 2 units of TaKaRa LA Taq DNA polymerase (purchased from TaKaRa Company). The PCR reaction conditions were: denaturation at 94°C for 1 min; 30 cycles at 94°C for 30 s, 63°C for 30 s, and 72°C for 1 min, and finally extension at 72°C for 5 min. The PCR product was TA cloned into the pMD19-T vector (purchased from TaKaRa Company), transfected with JM109 competent bacteria, and 5 clones were taken to extract plasmids, which were digested with Bcl I and HindIII respectively, and the plasmid containing the insert between t...

Embodiment 3

[0040] Example 3: Amplification of the full sequence of the chicken ovalbumin gene (OV).

[0041] Referring to GenBank J00895, the following primers were designed to amplify the entire sequence: primer 5, 29bp 5′AAC ATT TAC TGGGAA GCA CAT CTA TCA TC3′; primer 6, 28bp 5′GGA CTC TTG TTC AAC TTC TCA CCCACT A3′, and the product was 9016bp. The reaction system for PCR is 20μL, including: 50ng genomic DNA, 2μl 10×LA PCR buffer, 0.4mM dNTPs, 10μM primers, 2 units of TaKaRa LA Taq DNA polymerase (TaKaRa). The PCR reaction conditions were: denaturation at 94°C for 2 minutes; 35 cycles at 94°C for 30s, 69°C for 6 minutes, and finally extension at 72°C for 10 minutes. The PCR product was diluted 1000 times for use.

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Abstract

The invention discloses a gene targeting vector based on poultry adeno-associated virus, and a construction method thereof. The vector takes the poultry adeno-associated virus as a framework, wherein the inner sides of 5' and 3' LTR are connected with two homologous arms respectively; a gene component with positive selective resistance is connected between the two homologous arms; and two insulator elements are arranged between the gene component with positive selective resistance and the homologous arms respectively. The construction method comprises the following steps: (1) constructing a gene transfer vector of the poultry adeno-associated virus; (2) constructing pMD19-2HS4 plasmid; (3) amplifying OV complete sequence; (4) amplifying every component of the gene targeting vector and performing PCR splicing; and (5) constructing the gene targeting vector. The gene targeting vector has the advantages of effectively inhibiting position effect in a gene targeting process, improving the efficiency of screening positive clones, inhibiting random integration, improving the homologous recombination efficiency of the positive clones and having high gene transfer efficiency for non-active genes or temporal specific expressed genes.

Description

technical field [0001] The invention relates to a gene targeting vector and a construction method thereof, in particular to a gene targeting vector based on avian adeno-associated virus and a construction method thereof. Background technique [0002] Poultry has many advantages as a bioreactor: short generation interval, low feeding cost, high fertility; high protein content in poultry eggs, relatively simple ingredients, easy to purify; at the same time, there are natural protease inhibitors and maintenance in poultry eggs Systems in a sterile environment, such as lysozyme, etc., all of which effectively ensure the biological activity and stability of exogenous proteins; the existence of eggshells facilitates storage and transportation; and studies have shown that compared with some mammals, some proteins in poultry The glycosylation characteristics of the protein seem to be more similar to human proteins, which is very important to ensure the activity and application safet...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12R1/93
Inventor 徐世永刘红林
Owner JINLING INST OF TECH
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