Construction and application of PGRN and Rev-erbβ double genes knockout HEK293 cell lines / 生物工程学报

In order to study the molecular mechanism and physiological significance of the interaction between PGRN and Rev-erbβ, the PGRN gene in HEK293 (Rev-erbβ-/-) marked as C3-6 cell lines was knocked out by CRISPR/Cas9 system to generate the Rev-erbβ and PGRN double genes knockout HEK293 cell lines. First, four sgRNAs were designed for PGRN gene, and PGRN sgRNA2 and sgRNA3 with the higher activity were used to construct the Lentiviral vector, pLenti/CMV-Loxp-Cas9-sgRNA2-U6-sgRNA3-U6-Loxp-EF1α-Puro. Then, the lentivirus vector carrying Cas9 and double PGRN sgRNA were used to infect HEK293 C3-6 cells. Through drug screening, cloning and sequencing, we obtained the monoclonal HEK293 (Rev-erbβ-/-; PGRN-/-) marked as C3-6/23 cell lines. Using qRT-PCR and Western blotting, we detected PGRN mRNA and protein expression in C3-6/23 cell lines. Finally, genetic complementation was used to study the effect of PGRN-mediated Rev-erbβ on the regulation of the target gene promoter transcriptional activity in the C3-6/23 cell lines. In HEK293 C3-6/23 cell lines, the two DNA chains of PGRN gene were both deletion mutagenesis, and the expression mRNA and protein of PGRN did not reach the detection level. At the same time, the interaction between PGRN and Rev-erbβ enhanced the regulation of Rev-erbβ on the transcription of target gene promoter in the cell lines. Using CRISPR/Cas9 system, we successfully constructed the double knockout HEK293 (Rev-erbβ-/-; PGRN-/-) monoclonal cell lines. The study found that PGRN could affect Rev-erbβ on the regulation of target gene promoter transcription in the C3-6/23 cell lines; however, the mechanism of PGRN involvement in mediating Rev-erbβ in transcriptional regulation remains to be further studied.

Generation of islet β cell-specific Cre recombinase targeting vector by homologous recombination in bacteria / 第二军医大学学报

Objective To construct a knock-in targeting vector for expressing of Cre recombinase specifically in islet β cell and provide the key material for knock-in mice with Crr recombinase expressed in islet β cells, providing a knock-out animal model for studying the function of islet β cells. Methods In our study, we constructed a knock-in targeting vector using the third exon of insulin 2 (Ins2) as a target site with λ phage Red recombination system. Through a first homologous recombination, we cloned an about 12 kb genomic DNA fragment from the bacterial artificial chromosome (BAC) which contained Ins2 genomic DNA into a low copy vector pBR322-2s through gap repair. Meantime, a mini-targeting vector containing internal ribosome entry site (.IRES), DNA sequences encoding Crr recombinase and positive-negative-selection (PNS) gene was generated. After second recombination, the final Ins2-Cre targeting vector was generated. Results With the third exon of Ins2 used as the target, we successfully constructed the knock-in targeting vector expressing Cre recombinase which was controlled by endogenous Ins2 gene. Conclusion We have successfully constructed the knock-in targeting vector expressing Cre recombinase, it will provide an important material forcreating animal model of Cre recombinase which is specially expressed in islet β cells.

In vitro Efficacy of mda-7 Gene for Hepatocellular Carcinoma Gene Therapy Mediated by Human Ribosomal DNA Targeting Vector / 生物化学与生物物理进展

Human ribosomal DNA (hrDNA) targeting vector pHr is a homologous recombinant plasmid for human genome which developed in the State Key Laboratory of Medical Genetics. pHr was used to construct a recombinant plasmid pHr-CMG expressing mda-7/GFP fusion gene and its efficacy in the hepatocellular carcinoma cell line Bel-7402 was investigated. The expression of mda-7/GFP fusion gene was detected by fluorescent microscope, RT-PCR and Western blotting, and its function was detected by cell-cycle analyses, MTT assay and Hoechst33258 staining. The results demonstrated that pHr-CMG vector could express MDA-7/GFP fusion protein effectually and the mda-7 gene could induce cell apoptosis and proliferation suppression in Bel-7402 cell line, which might be caused by the G2/M cell cycle arrest. These results also suggested that human ribosomal DNA targeting vector system and the pHr-CMG vector may be applied in further gene therapy researches for hepatocellular carcinoma.

Studies on Gene Knocking Out of 3-Ketosteriod-1-Dehydrogenase in Mycobacterium neoaurum / 中国生物工程杂志

The selective side-chain cleavage of phytosterol to 4-androstene-3,17-dione(4-AD)and 1,4-androstadiene-3,17-dione(ADD)by Mycobacterium sp.was described.Because of the similarity in chemical structure between 4-AD and ADD,it is difficult to separate them from the fermentation broth.So far,it has been verified that the ADD can be produced by dehydrogenation of 4-AD.In this reaction,3-Ketosteriod-1-Dehydrogenase(ksdD)plays an important role.The gene knocking out method was used to solve the problem.Partial sequence of ksdD was obtained by PCR which was 631bp in length.Then,a targeting vector pUC19-MK was constructed,which was electroporate into the original strain Mycobacterium neoauru.The method of homologous recombination was used to knock out ksdD gene located in the chromosome of Mycobacterium neoauru.In this way,ksdD would lose its enzyme activity.In the result,5 transformants were screened.The experiments of steroid transformation by the transformants were carried out.The productivity of 4-AD reached 17.52% after 144h,which is 192% higher than the original strain.Meanwhile,the productivity of ADD reached 6.12%,which is 89.9% lower than the original strain.