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Method for genetic targeting of stem spermatogonium of poultry

A spermatogonial stem cell and gene targeting technology, which can be applied in microorganism-based methods, genetic engineering, plant genetic improvement, etc., can solve the problem of low proliferation efficiency of spermatogonial stem cells, improve the homologous recombination efficiency of positive clones, and achieve high protein yield. , the effect of short homology arms

Inactive Publication Date: 2009-11-11
JINLING INST OF TECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The second is the problem of targeting efficiency. Since the proliferation efficiency of spermatogonial stem cells is not high, effective targeting is very important

Method used

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  • Method for genetic targeting of stem spermatogonium of poultry
  • Method for genetic targeting of stem spermatogonium of poultry
  • Method for genetic targeting of stem spermatogonium of poultry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Construction of AAAV gene transfer vector (pAAAV-TR).

[0039] 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:

[0040] Primer 1: aa aagctt aaggatccatatgcggcc gctcgag aac agatct aGAGCTTCCTCGCTCACTGACTCG, the lowercase letters are the introduced multiple cloning sites, and the underlines indicate the HindIII, XhoI, and BglII sites in turn.

[0041] Primer 2: ttt gaattc The lowercase letters of tagACGTCAGGTGGCACTTTTC are the restriction restriction sites introduced, and the underline indicates the EcoR I site.

[0042] PCR is 20 μL reaction system including: 10 ng plasmid DNA, 2 μl 10×LA PCR buffer, 0.4 mM dNTPs, 10 μM primers, 2 units of TaKaRa LATaq DNA polymerase (purchased from TaKaRa Company). The PCR reaction conditions were: denaturation at 94°C for 2 minutes; 32 cycles at 94°C for 30s, 56°C for 30s, and 72°C...

Embodiment 2

[0044] Example 2: Amplification of HS4 and construction of pMD19-2HS4 plasmid.

[0045] Design primers according to chicken HS4 sequence (GenBank NW_001471556 293787-294082):

[0046] Primer 3: CCGCGGG TGATCA CGGGGAGAG is underlined to indicate the Bcl I site

[0047] Primer 4: TTCAGCCT AGATCT TTTTCCCCGTA is underlined to indicate the BglII site

[0048] The 20 μl reaction system for PCR includes: 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...

Embodiment 3

[0049] Example 3: Construction of chicken ovalbumin gene targeting vector.

[0050] Referring to GenBank J00895, the following primers were designed to amplify the full sequence of chicken ovalbumin gene: primer 5, 29bp 5′AAC ATT TAC TGG GAA GCA CAT CTA TCA TC 3′; primer 6, 28bp 5′GGA CTC TTGTTC AAC TTC TCA CCC ACT A 3 'The product is 9016bp. PCR is 20 μL reaction system including: 50ng genomic DNA, 2μl 10×LA PCR buffer, 0.4mM dNTPs, 10μM primers, 2 units of TaKaRa LATaq DNA polymerase (TaKaRa). The PCR reaction conditions were: denaturation at 94°C for 2min; 35 cycles at 94°C for 30s, 69°C for 6min, and finally extension at 72°C for 10min. The PCR product was diluted 1000 times for use.

[0051] See Table 1 for the design of PCR amplification primers and templates for each component of the gene targeting vector. (The pSUPER plasmid is a product of OligoEngine. For the pL-GFP plasmid construction method, see Journal of Nanjing Agricultural University 2008, 3(3): 97-101.)

...

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Abstract

The invention discloses a method for genetic targeting of stem spermatogonium of poultry. By using an adenovirus of poultry as a targeting vector, the method performs targeting operation on the stem spermatogonium of the poultry; the obtained genetically modified stem spermatogonium can be used for transplanting testis and obtaining a transgenetic gene; and by mating the normal poultry, the stem spermatogonium can be further used for breeding the transgenetic poultry. The method has the advantages that: the method can effectively realize the high-efficiency genetic targeting of the stem spermatogonium of the poultry; the used homologous arm is short, and the vector is easily established; and after the exogenous objective albumen is directly integrated in an albumin expression albumen high-efficiency promotor, the expressional specificity of the exogenous albumen of the transgenetic offspring is high, the yield of albumen is high. Moreover, the method also has the advantages of simple operation, safety, no pathogenicity, low immunogenicity and less cell toxins.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to a gene targeting method. Background technique [0002] Compared with mammalian bioreactors, avian oviduct bioreactors have many advantages, such as simple and efficient, low cost, easy purification, safety and stability, and have great development potential and market application prospects. The concept of poultry as a bioreactor experiment has existed for thirty years, but in fact in 2004 (lentiviral vector method to produce transgenic poultry appeared, McGrew M J, Sherman A, Ellard F M, et al.EMBO, 2004, 5: 728 -733) before, people focused more on how to achieve effective gene transfer in poultry. After the emergence of the lentiviral vector method, it included the development of other technical conditions. Now, people really have the conditions to think more about exogenous The specificity and high efficiency of protein expression, from a practical point of view, the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12R1/93
Inventor 徐世永刘红林
Owner JINLING INST OF TECH
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