Construction Of SLC3A2Lentiviral Vector And Transfection Of Sperm Cells

Background and Objective: Sperm-mediated gene transfer is simple to operate andless costly. The sperm has the natural featuers of spontaneous absorb foreign DNA, sothe exogenous gene maintain more integrity when it is transferred to sperm. Viralgenes can be integrated into the host cell genome and stably inherited. So lentiviralvectors become one of the main tools to produce transgenic animals. To investigatethe foreign gene into the sperm cell efficiency, sperm were incubated together withthe lentiviral vectors. Finally it is to filter out the best conditions.Method:1) Using genetic engineering techniques to target gene SLC3A2sequencefragment attached to the LV5vector. It is to recombinant vector construct. Then it isto complete the virus packaging, measured by quantitative PCR to calculate viraltiter.2) Lentiviral vectors containing the gene SLC3A2incubated with sperm cellsunder different conditions.1: Lentiviral vectors were incubated with the sperm cells atdifferent concentrations(104IU/ml，105IU/ml，106IU/ml).2: Sperm were incubatedwith lentiviral vectors is not the same length (1h，2h，3h，4h).3: Lentiviral vectorsand sperm are incubated at different temperatures(17℃，25℃，37℃).4: Lentiviralvectors with spermatozoa and incubated with different concentrations of Polybrene(0μg/mL，5μg/mL，10μg/mL，15μg/mL).3) Tested under different conditions, thelentiviral vector spermatozoa incubated efficiency of exogenous genes byimmunofluorescence and PCR.4) Tested under different conditions, the lentiviralvector spermatozoa incubated efficiency of sperm motility by microscopicexamination.Result:1) LV5-SLC3A2constructed recombinant vector by restriction enzymedigestion and sequencing report concluded vector was successfully constructed.2)Determination of the average titer of the lentiviral vector is2.03×108IU/ml.3) SLC3A2express higher in105IU/ml and106IU/ml. After2hours incubated, SLC3A2expressed. SLC3A2expressed at different temperatures. After incubation SLC3A2have highest expression by10μg/mL，15μg/mL Polybrene.4) sperm cell viability of3h,4h group was significantly lower than that of1h group. Sperm motility is higher in17℃. Sperm motility is lower, when sperm incubate15μg/mL Polybrene or106IU/mlvector.Conclusion:1) It is constructed SLC3A2lentiviral vector successfully.2) Sperm wereincubated with105IU/ml Lentiviral vectors and10μg/mL Polybrene2h at17℃, it ishave the best transfection efficiency.