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Method for obtaining transgenic chicken

A technology of transgenic chickens and exogenous genes, applied in the field of genetic engineering, can solve the problems of PGCs transgenic operation difficulties, destroy egg structure, sperm differentiation, etc., and achieve the effect of improving survival rate and hatching rate

Inactive Publication Date: 2006-08-23
YANGZHOU UNIV
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AI Technical Summary

Problems solved by technology

(2) Primordial germ cell (PGCs) transfection method: the number of available PGCs cells is small, the culture conditions are immature, and the transgenic operation of PGCs is difficult, which limits the development of this technology
(3) Blastodisk microinjection method: This method requires special instruments and equipment, and it is difficult to operate. During the microinjection, it will cause damage to the chicken blastodisc and destroy the structure of the egg, so it will appear during the incubation process. A large number of chicken embryos died
(4) Sperm-mediated method: The sperm-mediated exogenous gene transfer method is simple and easy to operate, has no damage to the pronucleus of the egg, conforms to the physiological fertilization process, and is easy to prepare in large quantities. However, the sperm has been differentiated and can only represent a single male individual. genetic information of the stage

Method used

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  • Method for obtaining transgenic chicken
  • Method for obtaining transgenic chicken
  • Method for obtaining transgenic chicken

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Experimental program
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Embodiment Construction

[0021] Step 1, pEGFP-N1 plasmid preparation

[0022] Transform Escherichia coli DH5α with pEGFP-N1, screen positive colonies with kanamycin (50 μg / ml), expand culture in LB medium, extract plasmid according to standard alkaline lysis method, take 1 μl of plasmid solution for agarose gel electrophoresis, quantify, and calculate extraction The concentration and total content of the plasmid in the solution were purified with a kit, the precipitate was washed with 70% alcohol, dried, and dissolved in sterile double distilled water, with a final concentration of 1 mg / ml. Single enzyme digestion was performed with Apal I restriction endonuclease at 37°C, and a fragment with a size of 4700 bp was recovered and purified by electrophoresis.

[0023] Step 2. Intra-testicular injection of plasmid DNA

[0024] Take 50 μg, 100 μg, 150 μg, and 200 μg of plasmid DNA, prepare cationic plasmid / polymer according to SofastTM instructions (vivo), and incubate at room temperature for 15-20 minute...

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Abstract

The method of obtaining transgenic chicken relates to gene engineering technology. Exogenous gene pEGFP-N1 plasmid is used to transfect stem spermatogonium, which is then differentiated inside body to obtain sperms carrying exogenous gene. Utilizing these sperms carrying exogenous gene can obtain great amount of transgenic descendents for improving chickení»s production performance, preserving chickení»s germ plasm resource, breeding disease variety, establishing disease mold, genetic treatment and development control, making bioreactor, and producing various kinds of bioactive medical protein hard to obtain in other methods.

Description

technical field [0001] The invention relates to the technical field of genetic engineering. technical background [0002] The more common methods for preparing transgenic chickens are: retrovirus infection, primordial germ cells (PGCs) transfection, blastoderm microinjection, and sperm-mediated methods. But these methods all have their own shortcomings. (1) Retroviral infection method: Because some viral vectors have the risk of causing tumors and causing viremia, the distribution of foreign DNA in various tissues is uneven, and it is difficult to integrate into germ cells, and the integration is often in multiple loci, so the offspring will have genetic variation. (2) Primordial germ cell (PGCs) transfection method: The number of available PGCs cells is small, the culture conditions are not mature, and the transgenic operation of PGCs is relatively difficult, which limits the development of this technology. (3) Blastodisk microinjection method: This method requires speci...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/70A01K67/027
Inventor 李碧春周冠月
Owner YANGZHOU UNIV
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