Production of Transgenic Pig Clone Embryo Expressed the Red Fluorescent Protein by Using the Somatic Cell Nucleus Transplantation Technology

[Objective] The research aimed to lay the foundation for producing the transgenic clone pig.[Method] The pig fetus fibroblast with the red fluorescent protein（RFP）gene that was transfected by the retrovirus was as the donor of nucleus transplantation.By using the somatic cell cloning technology,the development situation in vitro of clone embryo with RFP was studied.[Result] The fusion rate of RFP transgenic cell was 83.87% which had no significant difference with 80.56% of non-transgenic cell（P0.05）.The blastula rate in vitro of RFP transgenic somatic cell reconstructed embryo was 8.67% which had no significant difference with 6.56% of non-transgenic cell（P0.05）.After the reconstructed embryo of RFP transgenic somatic cell was transplanted into fifteen receptors,there was no conception individual.[Conclusion] The transgenic cell with the red fluorescent protein as the donor could successfully clone the transgenic embryo and obtain the transgenic blastula.

Screening and engineering of high-activity promoter elements through transcriptomics and red fluorescent protein visualization in Rhodobacter sphaeroides

The versatile photosyntheticα-proteobacterium Rhodobacter sphaeroides,has recently been extensively engineered as a novel microbial cell factory(MCF)to produce pharmaceuticals,nutraceuticals,commodity chemicals and even hydrogen.However,there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R.sphaeroides.In this study,several native promoters from R.sphaeroides JDW-710(JDW-710),an industrial strain producing high levels of co-enzyme Q10(Q10)were selected on the basis of transcriptomic analysis.These candidate promoters were then characterized by using gusA as a reporter gene.Two native promoters,Prsp_7571 and Prsp_6124,showed 620%and 800%higher activity,respectively,than the tac promoter,which has previously been used for gene overexpression in R.sphaeroides.In addition,a Prsp_7571-derived synthetic promoter library with strengths ranging from 54%to 3200%of that of the tac promoter,was created on the basis of visualization of red fluorescent protein(RFP)expression in R.sphaeroides.Finally,as a demonstration,the synthetic pathway of Q10 was modulated by the selected promoter T334*in JDW-710;the Q10 yield in shake-flasks increased 28%and the production reached 226 mg/L.These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R.sphaeroides-derived MCFs.

Construction of Expression Vector Capable of Excising Selectable Marker Gene

[ Objective] This study was to construct an expression vector capable of excising selectable marker gene, further eliminating the effect of marker gene on the functional study of target gene. [ Method] By using Cre/LoxP site-specific recombination system, DsRed2-1 vector was modified by introducing LoxP se- quence and multiple cloning site sequence, then the TK gene was ligated into this vector for negative selection. [ Results] The fragments introduced were recom- bined at the molecular level under induced condition, and the specific red fluorescence was also observed in the recombinant vector transfected cells at the cellular level. [ Conclusion] It is feasible to use this Cre/loxP system to excise marker genes, showing a broad application prospect.