Sleeping Beauty Transposon-mediated Efficient Gene Integration In Fish Cells

Sleeping Beauty(SB)transposon is an efficient genetic tool for boosting the efficiency of gene integration.In this study,the features of SB-mediated integration were examined in different fish cells.SB cis-configuration(cis)and trans-configuration(trans)systems carrying DsRed reporter gene were constructed to transfect black carp(Mylopharyngodon piceus)bladder cells(MPB)and grass carp(Ctenopharyngodon idellus)kidney cells(CIK)with different ratios of transposon versus transposase.The integration efficiency of different SB transposon systems was compared and the transposon targeted sites were characterized in fish genome.The SB transposition efficiency was also tested in Chinese sturgeon(Acipenser sinensis)muscle cells(ASM).Taken together,this study demonstrated that optimizing the ratio of SB transposon versus transposase increase the exogenous gene integration efficiency and generate mutant cells rapidly,providing theoretical basis for SB-mediated mutant library in fish cells.The results are as follows:(1)SB trans and cis transposon systems were constructed.The SB trans system consisted of SB transposon vector pT2 and SB transposase vector pSB(SB100X),the SB cis vector pT2_SB combined the transposase and transposon cassettes in one plasmid.The vcectors can both express puromycin-DsRed reporter element.(2)The transient transfection efficiency between SB cis and trans systems were compared.The MPB and CIK cells were transfected with various ratio of transposon versus transposase by Lipofectamine?2000: 1.cells transfected with pT2_SB(1 ?g);2.cells transfected with pT2(1 ?g);3.cells transfected with pT2(1 ?g): pSB=10:1;4.cells transfected with pT2(1 ?g): pSB=2:1;5.cells transfected with pT2(1 ?g): pSB=1:2?The expression of DsRed was observed under inverted fluorescence microscope and the transient transfection efficiency was calculated at 2 days post-transfection.In MPB cells,2:1 ratio of transposon versus transposase gave the highest transient transfection efficiency(10.8%)and the efficiency of SB cis vector was merely 3.6%.In CIK cells,10:1 ratio of transposon versus transposase gave the highest transient transfection efficiency(12.8%)and the efficiency of SB cis vector was about 8.3%.(3)The integration efficiency between SB cis and trans systems were compared.Transfected cells were subjected to 28 days of puromycin selection.The DsRed expression was detected by sqPCR,the relative DsRed expression level was examined by qPCR,and the percentages of DsRed positive cells were calculated by flow cytometry.In MPB cells,2:1 ratio of transposon versus transposase gave the highest DsRed expression level and the highest percentage of DsRed positive cells(81.6%),however,cells transfecting with SB cis vector suffered growth cessation.In CIK cells,1:2 ratio of transposon versus transposase gave the highest DsRed expression level and the highest percentage of DsRed positive cells(94.1%).These indicated that the integration efficiency of SB trans system was higher than SB cis system.(4)The SB transposon integration sequences were obtained by hiTAIL-PCR.It demonstrated that the SB transposon integration occurred randomly into TA dinucleotides with preference in fish genomes.(5)The integration efficiency of reporter gene was significantly increased by SB trans system in ASM cells.It suggested that the SB transposon system can be applied to various fish cells.