Construction and Evaluation of Lentiviral Vector pita-hbp1

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1 ISSN CN / Q Chinese Journal of Biochemistry and Molecular Biology ~ 1049 pita-hbp * HBP1 pita-hbp1 Western HBP1 Q78 Construction and Evaluation of Lentiviral Vector pita-hbp1 CHEN Yue-Ping 1 DU Yi-Cong 2 DONG Wei 3 WANG Wei-Bin 4 ZHANG Xiao-Wei 4 * 1 Department of Physiology and PathophysiologyPeking University Health Science CenterBeijing 2 High School Affiliated to Remin University of ChinaBeijing 3 First Hospital of Hebei Medical UniversityShijiazhuang and Molecular BiologyPeking University Health Science CenterBeijing China China China 4 Department of Biochemistry China Abstract Lentiviral vectors have been widely used in gene therapy and in laboratory biological study for their ability to deliver larger gene fragments into cells in the non-dividing phase In this studywe constructed a lentiviral vector pita-hbp1 expressing the HBP1 transcription factor and evaluated its infection efficiency of the packaged lentivirus by Western blot We found that both human tumor cells and normal cells were infected efficiently as determined by the expression of HBP1 Our results showed that HBP1 could be transduced exogenously into normal or cancer cells with our lentiviral system Key words lentiviral vector transcription factor HBP1 infection efficiency 1 RNA 9 gag pol env gag pol env 8 ~ 10 kb 1 2 tat rev 4 vif vpr vpu nef 2 DNA No * Tel xiaoweizhang@ bjmu edu cn Received May Accepted September Supported by General Program from National Natural Science Foundation of China No * Corresponding author Tel xiaoweizhang@ bjmu edu cn

2 11 pita-hbp CpG shrna ~ 7 RNAi 1 EF1 alpha 8 9 promoter UBC EF1 alpha RNA small hairpin RNA shrna shrna shrna HBP1 shrna plp1plp2plp3 293T shrna shrna RNA HBP1 Ⅲ RNA RNA Ⅲ RNA poly A RNA Ⅲ 4 5 T ~ 4 U U6 H1 RNA RNA HEK-293T Ⅲ ~ 21 nt RNA ~ 50 nt RNA stem loop shrna shrna plp1plp2plp3 Taq DNA polymerase 2 Mix Genstar shrna shrna BamH I Not I NEB RNA Ⅲ 4 ~ 5T T4 DNA DH5α 1 ~ 4 TIANGEN puromycin U 3 RIPA shrna shrna Genstar HA shrna Covance shrna pita pzsg promoter 1 2BS HeLa pita ECL BCA Pierce shrna 1 2 2BS293T HeLa shrna 10% FBS Hyclone DMEM Invitrogen 37 5% CO 2

3 HRP Taq DNA HBP1 1 h TBST 5 min 6 NC BamH I SuperSignal WestPico Chemiluminescent Not I HA Substrate PierceRockfordILUSA 1 ~ 5 min 30 s 5 min ATG TAA 12 TAG TGA PCR Lipofectamine 2000 Invitrogen HEK-293T 100 mm 10% DMEM puromycin 90% 1 h 1 ml DMEM 2 4 μg plp16 μg plp24 μg plp3 6 μg pita-hbp1 1 ml DMEM 2 1 pita 60 μl 5 min pzsg UBC 2 20 min 1 EF1 α EcoRI 5 h HindⅢ2 10% 1 5 EF1 α EF1 α 293T 24 h PCR 0 45 μm 1 1 V / EF1 α EF1 α PCR V 517 bp Fig 1B 8 ~ 12 h PCR EF1 α 24 h 2BS HeLa 1 puromycin 2BS HeLa 1 6 Western 3 ~ 5 RIPA 50 mmol / L Tris-HCL ph mol / L NaCl 0 1% Triton-X100 1 mmol / L EDTA50 mmol / L NaF1 mmol / L DTT 0 1 mmol / L Na 3 VO mmol / L PMSF 1 μg / ml leupeptin 1 μg / ml aprotinin 30 min r / min4 15 min 2 2 pita-hbp1 2 SDS 100 mmol / L Tris-HCl ph mmol / L DTT 4% SDS 0 01% 20% min NC TBST 20 mmol / L Tris-HCl ph mmol / L NaCl0 5% Tween 20 5 min 5% HBP1 BamHINot I 2 TBST 1h pita 0 2 ~ 1 μg / ml 4 TBST 5 min 6 2BS HeLa pita Fig 1A EcoRI HindⅢ PCR pzsg Fig 1C T4 EF1 α pzsg pita EF1 α pzsg Fig 1D 2 pita puromycin pita EGFP HBP1 HBP1 PCR

4 11 pita-hbp Fig 1 The lentiviral vector of pita was constructed by EF1α promoter inserted A The map of lentiviral vectors of pzsg and pita B PCR product of EF1α promoter PCR product was separated in 1% agarose electrophoresis The length of PCR product is 517 bp C UBC promoter was cut from the vector of pzsg pzsg was digested with EcoRI and HindⅢthen separated in 1% agarose electrophoresis D The construct of lentiviral vector pita pita was constructed by EF1α promoter inserted into pzsg pita was digested with EcoRI and HindⅢthen separated in 1% agarose electrophoresis HBP1 HBP1 PCR bp Fig 2A PCR HBP1 h BamHⅠNotⅠ EGFP HBP1 pita T4 D NA HBP1 pita pita-hbp1 pita-hbp1 30% puromycin HeLa 5 d Fig 2B 90% Fig 4 HBP1 plp1 plp2 plp3 pita BS HeLa 24 Fig 3

5 Fig 2 The lentiviral vector of pita-hbp1 was constructed by HBP1 cdna inserted into pita A PCR product of transcription factor HBP1 gene PCR product was separated in 1% agarose electrophoresis The length of PCR product is bp B HBP1 cdna were inserted into pita vector pita-hbp1 was digested with BamHI and NotIthen was separated in 1% agarose electrophoresis The fragment lengths of pita and HBP1 were bp and bprespectively Fig 3 Fluorescence detection for EGFP introduced into 2BS A B and HeLa cells C D by lentiviral vector pita before puromycin selection 100 Fluorescence detection for EGFP introduced into 2BS under white light A or fluorescence B Fluorescence detection for EGFP introduced into HeLa under white light C or fluorescence D Cells were transfected with pita- EGFP and then taken pictures under white light or fluorescence at day 1 after transfection The number of cells with EGFP expression were compared with that of cells without EGFP expression At least 300 cells were counted for each sample 2 4 HBP1 2BS HeLa puromycin 2BS 1 μg / Fig 4 Fluorescence detection for EGFP introduced into HeLa cells by lentiviral vector pita when puromycin selection for 5 days 100 Fluorescence detection for EGFP introduced into HeLa under white light A or fluorescence B Cells were transfected with pita-egfp and then taken pictures under white light or fluorescence at day 5 after puromycin selection The number of cells with EGFP expression compared with that of cells without EGFP expression At least 300 cells were counted for each sample mlhela 2 μg / ml SDS-PAGE Western pita- HBP1 Fig 5 40 μg p38 Loading control pita-hbp1 pita 1 HBP1 HBP1 72 kd HBP1 3

6 11 pita-hbp Fig 5 Transcription factor HBP1 can be efficiently expressed in 2BS cells A and 293T cells B by lentiviral vector pita Level of HBP1 was determined by Western blot analysis of 2BS and 293T cells infected with pita or pita-hbp1 p38 was used as loading control peptides J high-content screenj 10Heldt SARessler KJ pita HBP1 30% 30% HBP1 References 1 Tang HKuhen KLFlossie WS Lentivirus replication and regulationj Annu Rev Genet Asparuhova M BBarde ITrono Det al Development and characterization of a triple combination gene therapy vector inhibiting HIV-1 multiplication J J Gene Med Shizuko S Peptide nucleic acids as epigenetic inhibitors of HIV- 1J Int J Peptide Res Ther Sung MHSimon R Genomewide conserved epitope profiles of HIV-1 predicted by biophysical properties of MHC binding J Comput Biol Cockrell ASKafri T Gene delivery by lentivirus vectorsj Mol Biotechnol Kock NKasmieh RWeissleder Ret al Tumor therapy mediated by lentiviral expression of shbcl-2 and S-TRAILJ Neoplasia Delenda C Lentiviral vectors optimization of packaging transduction and gene expressionj Gene Med Rossi GRMautino MRMorgan RA High-efficiency lentiviral vector-mediated gene transfer into murine macrophages and activated splenic B lymphocytesj Hum Gene Ther Moffat JGrueneberg D AYang Xet al A lentiviral RNAi library for human and mouse genes applied to an arrayed viral Cell Amygdala-specific reduction of alpha1- GABAA receptors disrupts the anticonvulsantlocomotorand sedativebut not anxiolyticeffects of benzodiazepines in mice J J Neurosci Xu CYGu YHou WH et al Tetracysteine as a reporter for gene therapyj Biomed Environ Sci Zhang XKim JRuthazer Ret al The HBP1 transcriptional repressor participates in Ras- induced premature senescencej Mol Cell Biol Wu YBeddall MHMarsh JW Rev-dependent lentiviral expression vectorj Retrovirology Dull TZufferey RKelly Met al A third-generation lentivirus vector with a conditional packaging systemj J Virol Mautino MR Lentiviral vectors for gene therapy of HIV-1 infectionj Curr Gene Ther