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Method for preparing transgenic animal capable of carrying out specific expression of mammary gland from mammary gland epithelial cells

A technology for mammary epithelial cells and transgenic animals, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, and applications, can solve problems such as low efficiency, and achieve the effects of improving preparation efficiency, saving materials, and shortening development time.

Inactive Publication Date: 2012-02-22
YANGZHOU UNIV
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Problems solved by technology

However, due to the position effect of exogenous gene integration, only a part of these transgenic offspring have the function of expressing exogenous gene, so its efficiency is still low

Method used

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  • Method for preparing transgenic animal capable of carrying out specific expression of mammary gland from mammary gland epithelial cells
  • Method for preparing transgenic animal capable of carrying out specific expression of mammary gland from mammary gland epithelial cells
  • Method for preparing transgenic animal capable of carrying out specific expression of mammary gland from mammary gland epithelial cells

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Embodiment Construction

[0014] Experimental Materials

[0015] Culture medium DMEM / F12; fetal calf serum FCS; electroporation solution (Hypoosmolar Buffer) was purchased from eppendorf; FSH, LHRH and PG were purchased from Ningbo hormone factory; other involved chemical reagents can be purchased from Sigma company or other domestic bioengineering companies The company purchased a goat for experiments.

[0016] . Preparation of Human Lysozyme Transgenic Mammary Epithelial Cells

[0017] 2.1 Breast epithelial cell isolation and culture:

[0018] About 5 g of lactating goat mammary gland tissue was removed by aseptic surgery, and the tissue was cut into about 1 mm 3 Size, digest with type Ⅰ collagen / hyaluronidase digestion solution (containing type Ⅰ collagenase 200 IU / mL, hyaluronidase 100 IU / mL) and shake to digest, collect the cell suspension, wash 3 times with D-hank's, and count the cells And use culture medium (containing DMEM / F12, 10% FCS, sodium acetate 5 mmol / L, transferrin 5 μg / mL, ...

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Abstract

The invention relates to a method for preparing a transgenic animal capable of carrying out specific expression of a mammary gland from mammary gland epithelial cells. The method comprises the following steps of 1, transfecting mammary gland epithelial cells cultured in vitro with exogenous genes by expression vectors, and carrying out inducible expression by 3 to 8 micromole per liter of a prolactin, 2, carrying out screening to obtain a highly expressed mammary gland epithelial cell line, and 3, carrying out one-step nucleus transplant or two-step nucleus transplant of the highly expressed mammary gland epithelial cell line to obtain an individual capable of carrying out specific expression of a mammary gland. The method can improve transgenic animal preparation efficiency. Through the method, transgenic animal expression characteristics can be forecasted in a cell screening phase, and preparation efficiency of a transgenic animal capable of carrying out specific expression of a mammary gland is improved 3 times. Through an integrated position effect, an obtained transgenic individual has an expression probability of 10 to 30% and low-level expression may occur. Through the method, preparation of transgenic cells and identification of an expression potentiality of a prepared individual can be realized simultaneously, and thus more expression animals can be obtained.

Description

technical field [0001] The invention relates to animal mammary gland epithelial cell transgene technology; transgenic mammary gland epithelial cell induced expression technology and transgenic mammary gland epithelial cell nucleus transplantation technology. Background technique [0002] Cells transfected with exogenous genes in vitro can be used as nuclear donor cells for nuclear transplantation to obtain most of the animals integrating exogenous genes. Compared with traditional microinjection, the efficiency of this method has been significantly improved. However, due to the position effect of exogenous gene integration, only a part of these transgenic progeny have the function of expressing exogenous gene, so its efficiency is still low. The efficiency of transgenic animals will be further improved if cell lines with expression potential can be obtained before nuclear transfer. Mammary gland epithelial cells have the function of synthesizing and secreting milk, and are t...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/877A61D19/04A01K67/027
Inventor 成勇袁玉国
Owner YANGZHOU UNIV
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