Method for obtaining transgenic chicken by lentivirus vector mediated in vivo transfection of spermatogonial stem cell

A technology of lentiviral vectors and spermatogonial stem cells, applied in the field of obtaining transgenic chickens, can solve the problems of difficult acquisition and implantation of poultry eggs and fertilized eggs, few transgenic individuals, and limited acquisition methods, and achieve high production practical value and genome structure Improved, easy-to-operate results

Inactive Publication Date: 2009-08-12
刘红林 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The difficulties faced by other transgenic methods in poultry transgenesis are mainly manifested in the following aspects: 1. Due to the limitation of the special physiological mechanism of poultry (such as large yolk, eggshell, early development of fertilized eggs, etc.), poultry eggs, fertilized eggs, etc. The acquisition and implantation are extremely difficult, and at the same time, the male pronucleus in the fertilized egg is difficult to distinguish, and the method based on microinjection and nuclear transfer is difficult to operate
2. Restrictions on integration, subculture transmission and expression efficiency, such as retrovirus (except lentivirus)-mediated method, liposome-mediated method, sperm carrier method, etc., although individuals with exogenous gene integration can also be obtained, However, the efficiency is often low, it is difficult to pass on to the next generation, and the expression is weak or completely silenced
3. Due to the limitation of stem cell culture conditions and acquisition methods, due to the large differences with mammals, the stem cell culture system suitable for mammals is often not very satisfactory in poultry applications
[0007] 2. High cost and high virus consumption
[0009] 4. There are few transgenic individuals and slow population expansion

Method used

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  • Method for obtaining transgenic chicken by lentivirus vector mediated in vivo transfection of spermatogonial stem cell
  • Method for obtaining transgenic chicken by lentivirus vector mediated in vivo transfection of spermatogonial stem cell
  • Method for obtaining transgenic chicken by lentivirus vector mediated in vivo transfection of spermatogonial stem cell

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Example 1: Construction of a lentiviral vector plasmid expressing green fluorescent protein.

[0051] The enhanced green fluorescent protein gene (EGFP) comes from the peGFP plasmid (GeneBank Accession No: U55762, Clontech Company), which is obtained by separating and purifying the peGFP plasmid after Nde I and Xba I (NEB Company) double enzyme digestion and agarose gel electrophoresis . The pL-lacZ plasmid (Invitrogen) was digested with the same enzyme to obtain a DNA fragment containing the lentiviral transfer vector sequence, and the vector was dephosphorylated (NEB) with thermozyme phosphatase, separated and purified by agarose gel electrophoresis, and then purified with a quick ligation kit (LigaFastTM Rapid DNA Ligation System, Promega) was linked with EGFP, and finally transfected with JM109 competent bacteria to amplify the plasmid, and the new plasmid was named pL-EGFP ( figure 1 ).

[0052] Analyzed by Gene Construction Kit 2.0 software, the pL-EGFP plasmid ...

Embodiment 2

[0053] Example 2: Construction of a lentiviral vector plasmid expressing human tissue plasminogen activator (tPA) green fluorescent protein fusion protein.

[0054] In order to insert tPA into the viral vector, primers were designed based on the tPA sequence to introduce Xho I and BamH I restriction sites before and after the tPA coding region. The forward primer introduced four site mutations before the start codon ATG, and the change of +3 position from A to G did not change the conserved sequence of Kozak transcription initiation and did not affect the transcription efficiency of tPA in eukaryotic cells. The reverse primer introduces three mutation sites before the stop codon, which will result in the deletion of the last three amino acids after digestion and fusion with the EGFP gene.

[0055] Primer 1: GCGGGAATT CTCGAG ATGGATGCAA is underlined to indicate the Xho I restriction site

[0056] Primer 2: GATTATCAC GGATCC ATGTTGTCAC is underlined to indicate the restricti...

Embodiment 3

[0060] Example 3: Production of virus.

[0061] 1) One day before transfection, digest 293FT cells with trypsin, and take about 6×10 6 Cells are planted at 75cm 2 Add about 10ml of complete medium to the culture bottle for culture.

[0062] 2) On the day of transfection, when the cells reach 90-95% confluence, remove the culture medium and add 6ml of 293FT medium without double antibodies.

[0063] 3) Prepare DNA-liposome transfection complex:

[0064] A: Take 11.25 μg of virus-assisted packaging plasmid mixture (invitrogen) and 3.75 μg of pL-EGFP or pL-tPAGFP plasmid, a total of 15 μg of DNA, and add it to 1.9mL 293FT serum-free medium without double antibodies, mix gently, and set aside.

[0065] B: Take another centrifuge tube and add 45 μL of liposome Lipofectamine TM 2000 (invitrogen) into 1.9mL 293FT serum-free and double-antibody-free medium, gently blow and aspirate to mix, and incubate at room temperature for 2-4min (no more than 5min).

[0066] Quickly mix with ...

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Abstract

The invention discloses a method for obtaining transgenic chickens through spermatogonial-stem-cell in vivo transfection mediated by lentivirus vectors. The method comprises the steps of directly injecting a lentivirus vector into a testis of a cock, transferring genes of endogenous spermatogonial stem cells to generate transgenic sperms, mating the cock with a normal hen and then obtaining the transgenic chickens. Through the way with the endogenous spermatogonial stem cells, the method for obtaining transgenic chickens through spermatogonial-stem-cell in vivo transfection mediated by lentivirus vectors can avoid opening poultry eggs, and has the advantages of low virus consumption, fast group extension and capability of stable passage.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for obtaining transgenic chickens. Background technique [0002] Although the concept of transgenic poultry research has been proposed for nearly 30 years, the progress has been unsatisfactory due to the lack of effective means of introducing foreign genes. In 2002, Lois et al. (Lois C, Hong E J, Pease S, et al.Science, 2002, 295: 868-872) used lentiviral vectors to produce high-efficiency integrated transgenic mice. The experimental results gave important hints to the production of transgenic poultry . In 2004, McGrew et al. (McGrew M J, Sherman A, Ellard F M, et al. EMBO, 2004, 5: 728-733) used similar viral vectors to directly inject fresh chicken embryos to obtain highly efficient germline transgenic chickens, and G0 integrated transgenes The efficiency is as high as 70%, and no transgene silencing phenomenon is found in the subculture. The...

Claims

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Application Information

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IPC IPC(8): C12N15/867A01K67/027C12R1/93
Inventor 徐世永刘红林
Owner 刘红林
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