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[Construction of HBD-3 gene mammary-specific expression vector and eukaryotic expression]
Wei Peng1, Zhigang Lan, Jingjing Ma
1Institute of Biotechnology, College of Animal Veterinary Medicine, Northwest Agiculture and Forestry University, Yangling 712100, China.
Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology
|October 20, 2009
Summary
Researchers created genetically modified bovine cells capable of producing transgenic animals. This advancement in somatic cell nuclear transfer (SCNT) aims to improve the efficiency of producing cloned transgenic cattle.
Area of Science:
- Animal Biotechnology
- Gene Engineering
- Reproductive Biology
Background:
- Somatic cell nuclear transfer (SCNT) is a key technology for producing transgenic animals.
- Developing efficient methods for generating competent donor cells is crucial for SCNT success.
- Mammary-specific expression systems offer potential for targeted gene delivery.
Purpose of the Study:
- To establish human beta-defensin-3 gene transgenic cell lines as competent donor cells for SCNT.
- To construct a mammary-specific expression vector for gene transfer.
- To improve the efficiency of transgenic animal production.
Main Methods:
- Obtained human beta-defensin-3 (hBD) via RT-PCR and cloned it into expression vectors.
- Constructed the mammary-specific expression vector pEBCD.
- Transfected Holstein fetal fibroblast cells with pEBCD using Lipofectamine TM-2000.
- Selected and identified stable transfectants using G418, PCR, RT-PCR, and EGFP detection.
- Confirmed hBD expression in transfected cells via Western blotting.
Main Results:
- Successfully constructed the mammary-specific expression vector pEBCD.
- Generated stable G418-resistant transgenic fibroblast cell lines.
- Confirmed stable integration of the human beta-defensin-3 gene into the host cell chromatin.
- Detected expression of human beta-defensin-3 in the supernatant of stable transfected mammary epithelial cells.
Conclusions:
- Established transgenic cell lines expressing human beta-defensin-3 are competent donor cells for SCNT.
- This method provides a foundation for improving transgenic cloning efficiency.
- The developed vector and cell lines hold promise for producing genetically engineered animals.
