Production of artificial synthetic spidroin gene 4S-transgenic cloned sheep embryos using somatic cell nuclear transfer

Abstract

Spider silk, which has remarkable characteristics, has wide application prospects in many fields. Many researchers have explored potential methods for directly producing spider silk proteins and spidroins with mechanical properties or obtaining recombinant spider silk fibers by genetic engineering methods. However, there are still some shortcomings with these methods, such as inability to simulate the fibrosis process of spider silk. In this study, a high glycine/tyrosine protein gene (HGT) promoter originate from sheep was first cloned by PCR. The HGT promoter was ligated into pcDNA3.1 and pcDNA3.1-HGT was obtained. After linking with the synthesized and polymerized gene 4S, a eukaryotic expression vector pcDNA3.1-HGT-4S was constructed using a series of molecular methods. Sheep fibroblasts transfected with the linearized plasmid using a liposome-mediated method were screened with G418 and a transgenic cell line was established. Cells from the transgenic line were used as nuclear donors to construct embryos with somatic cell nuclear transfer (SCNT). Reconstructed embryos derived from transgenic cells were able to develop in vitro successfully. PCR was carried out and results demonstrated that the synthetic spidroin gene 4S had integrated into the embryo genome. In summary, we explored a method and successfully obtained artificial synthetic spidroin gene transgenic sheep cloned embryos with a hair follicle specific promoter by SCNT. Further research is necessary on transgenic sheep with synthetic spidroin genes expressed in hair follicles.

Keywords:

• Spidroin gene
• sheep
• SCNT
• cloned embryo

Ethical approval

The study was approved by the Laboratory Animal Management and Ethics Committee of Northeast Forestry University, Harbin, China. All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals. Efforts were made to minimize suffering.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported financially by the Fundamental Research Funds for the Central Universities [2572019BD02] and National Natural Science Foundation Project of China [31000990].