PCR. A Novel Method for the Determination of Recombinant Lentiviral Titer and Infectivity by qrt-pcr. MA Hai-yan FANG Yu-dan ZHANG Jing-zhi *

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1 13 5 Vol13 No Life Science Research Oct *,, :,, GFP, GFP, : ; ; ; ; : Q331 :A : (2009) A Novel Method for the Determination of Recombinant Lentiviral Titer and Infectivity by qrt- MA Hai-yan FANG Yu-dan ZHANG Jing-zhi * School of Medicine Shanghai Jiaotong University Shanghai Children s Hospital Shanghai Institute of Medical Genetics Shanghai China Abstract Lentiviral vector is being widely used in the study of gene therapy in animal models and in generating transgenic animals However determination of lentiviral particles and their infectivity is essential before their being used Such a requirement can be accurately achieved by qrt- Refered by infectious units got from GFP reporter assay it showed a positive correlation between the two approaches A reliable accurate and rapid method is therefore established for the determination of the recombinant lentiviral titer and the infectivity Key words lentiviral vector qrt- viral titer integration copy number infectivity Life Science Research ~398 [1~3] [4] 8 kb [5] HIV-1 MuLV 5 [6] : : : 2007AA ZR : * Tel jzhang38@hotmailcom

2 5 : μl DNase 5 μl 10 DNase I Buffer 2 μl DNase I GFP 5 U / ul Takara Bio Inc Shiga Japan 2 μl p24 - ELISA RNase inhabitor Takara Bio Inc Shiga Japan DEPC 50 μl min DNase 350 μl STE 20 μl 10% SDS 5 μl K 20 g / L AMRESCO Solon OH min 20 μl DEPC 14 LTR RNA / LTR DNase I DNA 1 μg RNA 20 pmol RT- GFP 5 -GAGAGCTCCCAGGCTCAGATC-3 2 μl 5 RT Buffer 1 μl MLV Takara Bio Inc Shiga Japan 10 μl 37 1 h 15 LTR nmol / L 250 nmol / L 25 μl 10 Ex-Buffer 11 3 FUGW 5 μl 25 μl egfp 89 Corbett Life Science RG-3000 Sidney VSVG Australia 95 5 min s Marine Medical Center Research Institute Promega ProFection Kit FUGW 15 μg μg VSVG 75 μg 6 h 10% μl FBS DMEM 37 5% CO 2 60 h 13 RNA 15 μmol Mg μmol dntp 1 U ExTaqE s nm 293T ATCC American Type Culture Collection 1 ProFection Promega Table 1 The sequences of primer and probe of Realtime FBS Hanks Salmon Sperm DNA Primer Sequences Gibco BRL STE 10 mmol / L Tris ph LTR-F 5 -ACAGCCGCCTAGCATTTCAT mmol / L EDTA ph mol / L NaCl LTR-P 5 -GAGAGCTCCCAGGCTCAGATC-3 12 FUGW LTR-Probe 5 -ACATGGCCCGAGAGCTGCATCC-3 293T 70% 16 FUGW 293T g mg / L Polybrene 15 h Sigma-Aldrich Inc St Louis MO Hanks T / 37 2 h 2 d

3 DNA 2 d ml EP tube 200 g 5 min LTR 200 μl STE 20 μl 10% SDS 10 μl K 37 4 h LTR g 12 min LTR g / ml 6 min 1/10 3 mol/ml 1 2 NaAc h / ml g 4 20 min 100 μl TE T DNA LTR 15 GFP Norm fluore 10^-1 10^-2 Threshold Threshold cycle R= R^2= Efficiency=096 M=0293 B= ^ Cycle number 1 Fig1 Fluorescence intensity curve of Real-time 0 10^4 10^5 10^6 10^7 10^8 10^9 Concentration Copy number 2 Fig2 Standard curve of Real-time 22, =LTR / ± IU / ml GFP μl μl 293T 2 d 2 d DNA LTR GFP μl 0001 μl GFP % 6% 05% = GFP R = 099 R 2 = 099 Efficiency = ± IU/mL 24 GFP DNA, LTR GFP

4 5 : GFP GFP A B C 3 293T GFP (A) 01 μl ; (B) 001 μl ; (C) 0001 μl Fig3 GFP expression in infected 293T cells after 10 fold dilution A 293T cells infected by 01 μl virus B 293T cells infected by 001 μl virus C 293T cells infected by 0001 μl virus 2 GFP Table 2 Comparison of lentivirus titer determined by GFP reporter assay and qrt- approach Dose of viral particles Percentage of GFP + cells 520% 60% 05% GFP reporter assay Infectious units qrt- assay Infectious units T Notes 293T cells in each well are: [8~10] 25 = / 100% 265 ± 107% 3 [11,12] [13] [14] HIV-1 HIV-1 1 p24 p24 ELISA ELISA [7] HIV-1 2 3

5 LTR : (References): 1 [1] WU X LI Y CRISE B et al Transcription start regions in the human genome are favored targets for MLV integration[j] Science [2] DEPALMA M MONTINI E SANTONIDESIO F R et al LTR 2 GFP hematopoietic cells[j] Blood GFP hotspots[j] Cell GFP 3 Neurosci Res GFP [J] Jing-zhi GUO Xin-bin XIE Shu-yang et al Promoter trapping reveals significant differences in integration site selection between MLV and HIV vectors in primary [3] SCHRODER A R SHINN P CHEN H et al HIV-1 integration in the human genome favors active genes and local [4] JAKOBSSON J ERICSON C JANSSON M et al Targeted transgene expression in rat brain using lentiviral vectors[j] [5] Science cells[j] RNA [7] LIU Yin transgenic animal development[j] Int J Genet lentiviral vectors[j] Science T 4% embryonic stem cells and preimpalntation embryos[j] Natl Acad Sci U S A EMBO Reports ZHANG Production of transgenic mice carrying green fluorescence protein gene by a lentiviral vector-mediated approach[j] Progress in Nature [6] STEWART S A OYLOCHOOM D M PALLISER D et al Lentivims-delivered stable gene silencing by RNAi in primary [J] Application of lentiviral vectors in [8] MAY C RIVELLA S CALLEGARI J et al Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin[j] Nature [9] HAN X D LIN C CHANG J et al Fetal gene therapy of α-thalassemia in a mouse model[j] PNAS [10] LI W XIE S Y GUO X B et al A novel transgenic mouse model produced from lentiviral germline integration for the study of β-thalassemia gene therapy[j] Haematologica 2008 [11] LOIS C HONG E J PEASE S et al Germline transmission and tissuespecific expression of transgenes delivered by [12] PFEIFER A IKAWA M DAYN Y et al Transgenesis by lentiviral vectors lack of gene silencing in mammnlian Proc [13] HOFMANN A KESSLER B EWERLING S et al Efficient transgenesis in farm animals by lentiviral emtovora vectors[j] [14] HOFMANN A ZAKHARTCHENKO V WEPPERT M et al Generation of transgenic cattle by lentiviral gene transfer into oocytes[j] Biol Reprod