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Method for acquiring animal permanent transgenes through spermatogonial stem cells (SSCs), recombinant plasmid and germ cells of permanent transgenes

A technology of spermatogonial stem cells and recombinant plasmids, applied in the field of recombinant plasmids and permanently transgenic germ cells, can solve the problems of high cost, low efficiency, hindering the application of transgenic technology, etc., and achieve the effect of convenient application

Inactive Publication Date: 2011-10-12
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some transgenic technologies have higher efficiency in transgenic cells or embryos under in vitro conditions, but when these transgenic technologies are transgenic in vivo or produce transgenic offspring, the efficiency of these transgenic technologies is greatly reduced because the internal environment is difficult to change with the needs of transgenics
For example, the overall efficiency of gene transfer through nuclear transfer is less than 1%, making it difficult to popularize and apply
Moreover, when these technologies are used for large animals, the operation is more complicated and difficult, the cost is higher, and the efficiency is lower.
The low transgenic efficiency in large animals seriously hinders the application of transgenic technology in these animals

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Cloning of target gene oIGF-1

[0023] 1. Extraction of RNA

[0024] 1. Take the tissue (about 10g) and immediately immerse it in 1mL Trizol. The sample volume generally does not exceed 10% of the Trizol volume. Homogenize the tissue with a tissue homogenizer.

[0025] 2. Add 0.2ml of chloroform, shake vigorously by hand for 15-30s to mix well, and let stand at room temperature for 5-10 minutes.

[0026] 3. Centrifuge at 12000rp / min for 15min. The sample will be divided into three layers: the lower organic phase, the middle layer and the upper colorless water phase. RNA mainly exists in the water phase. Move the water phase (do not absorb any substance in the middle layer) to In a new 1.5mL microvolume ep tube;

[0027] 4. Add an equal volume of isopropanol and let stand at room temperature for 10 minutes.

[0028] 5. Centrifuge at 12,000rp / min for 10min, discard the supernatant, add 1ml of 75% ethanol and shake to mix.

[0029] 6. Centrifuge at 12,000...

Embodiment 6

[0088] Embodiment 6: Transgenic Detection - Amplification of the Gene of Interest

[0089] 1. DNA amplification: using the extracted DNA as a template, the kit instructions amplify the target gene. PCR reaction system: Add in sequence to the sterilized EP tube:

[0090] 10×PCR buffer 5μL

[0091] dNTP Mixture 1μL

[0092] MgCl2 (25mM) 3μL

[0093] Plasmid template 2 μL

[0094] FP 1μL

[0095] RP 1μL

[0096] Taq DNA polymerase 0.5 μL

[0097] Add sterilized deionized water to make up to a total volume of 50 μL. The reaction conditions were as follows: 95°C for 4 min; 35 cycles of 94°C for 35 s, 56°C for 50 s, and 72°C for 1 min; and an extension at 72°C for 10 min. After the reaction, the product was identified by 1.0% agarose gel electrophoresis for PCR results.

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PUM

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Abstract

The invention discloses a method for acquiring animal permanent transgenes through spermatogonial stem cells (SSCs), recombinant plasmid and germ cells of the permanent transgenes, and belongs to the technical field of gene engineering. The method for acquiring the animal permanent transgenes through the SSCs comprises the following steps of: (1) amplifying a target gene fragment; (2) connecting the amplified target gene fragment and a Lenti-GFP-T2A vector to obtain the recombinant plasmid; (3) efficiently packing the recombinant plasmid of the step (2) in 293T cells; and (4) injecting the external packed plasmid of the step (3) into a convoluted tubule and other parts of a testicle respectively to obtain the germ cells of the transgenes. The invention has the advantages that: the transgene efficiency is ultrahigh, and the overall transgene efficiency can reach over 90 percent; transgenic sperms can be continuously generated; the transgenic sperms are convenient to use; and stable; and permanent transgenes can be formed.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for obtaining permanent transgenes of animals through spermatogonial stem cells, recombinant plasmids and permanently transgenic germ cells. Background technique [0002] Genetically modified large animals have broad application prospects and can directly serve agriculture, animal breeding, and biomedicine—production of human medical proteins, humanized organs, etc. Therefore, in recent years, a variety of transgenic technologies have come out, for example, the pronuclear injection technology that is traditionally mainly used for mouse transgenes, and later transgenic transgenes through nuclear transfer, transgenic electroporation transgenes through physical and chemical methods, liposome transgenes, transgenes using Viral vector transgene with high transfection rate. [0003] The use of zinc-finger endonucleases and transposons transgenes has re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/63C12N5/10
Inventor 曹文广
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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