title: Construction and expression of lentiviral vectors encoding recombinant mouse CREBZF in NIH 3T3 cells.
abstract: CREBZF, also known as Zhangfei or SMILE, is a member of the CREB/ATF protein family. CREBZF has mainly been considered as a basic region-leucine zipper transcription factor that functions in coordination with other transcription factors and plays a role in latent HSV-1 infection, apoptosis and the mammalian endoplasmic reticulum stress and unfolded protein response. In this study, we constructed recombinant lentiviral vectors for CREBZF short hairpin RNA (shRNA) expression and over-expression to improve understanding of the mechanisms regulating CREBZF. The CREBZF ORF sequence was cloned into the lentiviral shuttle plasmid pCD513B-1, and various shRNA oligonucleotides and one negative control (shN) were cloned into the pCD513B-U6 expression vector. The recombinant lentivirus was packaged and transduced into NIH 3T3 cells. CREBZF mRNA and protein expression were examined using real-time reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting, respectively. The over-expression vector and the most effective shRNA vector significantly affected the expression of CREBZF mRNA and protein. Both of the CREBZF recombinant lentiviral vectors were successfully constructed. The over-expression vector significantly increased the expression of exogenous CREBZF and inhibited the growth of NIH 3T3 cells compared to controls. The most effective shRNA lentiviral vector, pCD513B-U6-CREBZF-shRNA-3, was transformed, leading to significant knockdown of the CREBZF gene. We conclude that CREBZF the recombinant lentiviral vectors are promising tools for regulating the expression of CREBZF in NIH 3T3 cells.
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China; College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Fenglei
authors.initials: F
authors.lastname: Chen
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China; College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Peng Fei
authors.initials: PF
authors.lastname: Lin
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China; College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China; College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Sun
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China; College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Enqi
authors.initials: E
authors.lastname: Du
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Aihua
authors.initials: A
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China; College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China. Electronic address: yapingjin@163.com.
authors.forename: Ya Ping
authors.initials: YP
authors.lastname: Jin
mesh_terms: D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D049109:Cell Proliferation / Q000235:genetics; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D041681:NIH 3T3 Cells; D034741:RNA, Small Interfering; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C570109:SMILE protein, mouse
keywords: CREBZF; Gene over-expression; Lentivirus; Mouse; shRNA