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Recombinant bovine fetal fibroblast containing hBD3 mammary gland specific expression vector

A technology of fibroblasts and expression vectors, applied to cells modified by introducing foreign genetic materials, recombinant DNA technology, using vectors to introduce foreign genetic materials, etc., can solve problems such as cell lines without nuclear donor cells

Inactive Publication Date: 2011-09-07
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, using hBD3 as the target gene and using animal cells as the host cell, especially the somatic cells of cows as the expression vector of the host cell, has not been reported; nor has hBD3 been used as the target gene to construct the nuclear donor cell of the gene cloned embryo department report

Method used

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  • Recombinant bovine fetal fibroblast containing hBD3 mammary gland specific expression vector
  • Recombinant bovine fetal fibroblast containing hBD3 mammary gland specific expression vector
  • Recombinant bovine fetal fibroblast containing hBD3 mammary gland specific expression vector

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Embodiment Construction

[0032]The present invention first constructs the mammary gland-specific expression vector pEBB containing hBD3 and green fluorescent protein (EGFP) genes, and then uses the liposome method to introduce the exogenous expression vector pEBB into bovine fetal fibroblasts, and observes the expression of the marker gene EGFP by a fluorescence microscope. The positive cells were screened by G418 and identified by PCR to confirm the integration of the target gene hBD3 into the genome of bovine fetal fibroblasts; finally, the bovine fetal fibroblasts transfected with hBD3 gene were transplanted into bovine enucleated cells as nuclear donors From the oocytes, the transgenic cloned embryos were obtained and identified by PCR. The transgenic cloned embryos were transferred into the uterus of 64 recipient cows, and 21 transgenic cloned embryo recipient cows with 3 months of pregnancy were obtained.

[0033] The specific reagents involved are as follows: G418, DMEM medium and LipofectamineT...

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Abstract

The invention relates to a bovine fetal fibroblast containing an hBD3 mammary gland specific expression vector. The bovine fetal fibroblast comprises an hBD3 mammary gland specific expression vector containing a target gene hBD3, wherein a regulating element is inserted in the 5'end and the 3'end of the hBD3 gene respectively; and the hBD3 mammary gland specific expression vector is a pEBB expression vector. A host cell of the bovine fetal fibroblast containing the target gene hBD3 is a bovine fetal fibroblast, and integrates the target gene hBD3 into a genome of the bovine fetal fibroblast through transfecting the exogenetic expression vector pEBB. Transgenic cloned embryos can be obtained through SCNT by using the bovine fetal fibroblast containing the target gene hBD3 as a core donor cell for constructing the transgenic cloned embryos by nuclear transfer, so that a cow with a trans-hBD3 gene is expected to be produced by transferring the embryos into a uterus of a recipient bovine.

Description

technical field [0001] The invention belongs to the technical field of transgenic cloning animals, and relates to the construction of nuclear donor cells of transgenic cloned embryos, in particular to a bovine fetal fibroblast containing hBD3 mammary gland-specific expression vector. Background technique [0002] Mastitis is a very common disease in dairy cows. This inflammation is caused by a variety of pathogenic microorganisms, mainly Escherichia coli (E.coli) and Staphylococcus (S.aureus) (NOTEBAERT S, DEMON D, VANDEN BERGHE T et al.Inflammatory mediators in Escherichia coli-inducedmastitis in mice[J].Comparative Immunology, Microbiology and Infectious Diseases, 2008, 31(6):551-565; ZHEN YH, JIN LJ, LI XY et al.Efficacy of specific egg yolk immunoglobulin (IgY) to bovine mastitis caused by Staphylococcus aureus [J]. Veterinary Microbiology, 2009, 133(4): 317-322.). Mastitis has caused huge economic losses to the dairy industry. At present, the treatment of mastitis is s...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N15/06
Inventor 华松张涌马晶晶权富生王勇胜刘军郑月茂
Owner NORTHWEST A & F UNIV