Transgene method of reverse transcription of transfection primitive spermatogonium abduction in virion

A retrovirus, spermatogonial stem cell technology, applied in other methods of inserting foreign genetic materials, genetic engineering, plant genetic improvement, etc., to achieve the effect of simple operation and low investment cost

Inactive Publication Date: 2003-10-08
MILITARY VETERINARY INST MILITARY SUPPIES PLA
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  • Application Information

AI Technical Summary

Problems solved by technology

In 1994, someone carried out xenotransplantation of spermatogonial stem cells (Brinster RL.And Avarbock MR.PNAS, 1994), and proposed the possibility of culturing spermatogonial stem cells in vitro to achieve germline modification, but so far there has been no successful report

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Taking LacZ (beta-galactosidase) as the target gene, it was cloned into the downstream of the 5' long terminal repeat (5'-LTR) promoter of the recombinant retroviral vector plasmid pLXSN, and constructed into a packaging vector. The packaging cells PA317 were transfected, and after screening and proliferation, a toxin-producing cell line was obtained, and the supernatant of the cells was collected to concentrate the virus by ultracentrifugation. Dissolve the concentrated virus in phosphate buffer solution (PBS, pH7.2) containing 0.05% (w / v) trypan blue, 0.008g / L Polybrene, operate under a stereoscopic mirror, and inject it for 13-17 days In the seminiferous tubules of young mice. Mice were injected for mating 40 days postoperatively. Take offspring mouse tissues for DNA integration detection (polymerase chain reaction (PCR) uses primers from the nucleic acid sequence of the target gene, and a single nucleotide is catalyzed by a heat-resistant DNA polymerase to elongate...

Embodiment 2

[0019] Taking tPA (tissue plasminogen activator) as the target gene, it was cloned into the 5-LTR of the recombinant retroviral vector pLXSN and downstream of the CMV promoter of pLNCX, and then constructed into a packaging vector, which was transfected into PA317 cells. After screening and proliferation, a toxin-producing cell line is obtained, and the supernatant of the cells is collected to concentrate the virus by ultracentrifugation. Dissolve the concentrated virus in PBS (pH7.2) containing 0.05% (w / v) trypan blue, 0.008g / L Polybrene, operate under a stereoscopic mirror, and inject it into 14-16 day old mice. Inside the seminiferous tubule. Mice were injected for mating 40 days after surgery. The offspring mouse tail tissue was used for DNA integration detection (PCR, Southern hybridization), embryonic fibroblast culture was used for expression detection (RT-PCR), and passage test was carried out. result:

[0020] The toxin-producing cell line transfected with the reco...

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Abstract

A transgenic method using recombinant retrovirus as mediation to transfect stem spermatogonium in body includes cloning target gene to retrovirus carrier, transforming PA317 cells, collecting and concentrating the recombinant virus generated by clone of positive cells, injecting it in the contorted seminiferous tubule of mouse, and reproducing filial generation. Its advantages are simple operation and high repeatability.

Description

Technical field: [0001] The invention relates to a transgenic method, in particular to a transgenic method for in vivo transfection of spermatogonial stem cells mediated by a recombinant retrovirus. Background technique: [0002] Mammalian transgenic methods that have been established and applied include embryonic microinjection, embryonic stem cell-mediated and recombinant retrovirus-mediated; the first is the microinjection technology of fertilized eggs, that is, the target gene fragments are directly introduced into In the male pronucleus of the fertilized egg, the advantage of this technique is that there is no limit to the size of the gene fragment, and the length can reach hundreds of kb. The introduction process of the target gene is relatively intuitive. The disadvantage is that the instrument is expensive, the operation is complicated, and the investment rate is high. Low; the second is embryonic stem cell-mediated transgenic technology, embryonic stem cells are plu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N15/867
Inventor 扈荣良张守峰王凤阳
Owner MILITARY VETERINARY INST MILITARY SUPPIES PLA
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