A Preliminary Study On Lentiviral Mediated HLF Transgenic Chicken

Transgenic chicken is an important economic animal and chicken oviduct bioreactor is an ideal bioreactor for producing pharmaceutical proteins in the livestock industry.Transgenic chicken production has a broad application prospect.In this study,we constructed different lentiviral vectors and explore the methods that the production of transgenic chicken.The results are as follows:Construction of lentiviral vector:We construct overexpressed vector is that GV358-LV-HLF,pLVX-IRES-LTF-EGFP and pCDH-CMV-LTF-mcherry lentiviral vector in this study.Three vector HLF and LTF gene sequences are regulated with CMV promoter.Lentiviral was packaged by the three plasmid packaging lentivirus system in our laboratory.The titer of lentivirus was detected by the method of double dilution method,which was 109TU/mL.We explore the different factors about the efficiency lentivirus transgenic produced:our study explored the effects of different factors on hatching rate of eggs.The different way of sealing test results showed that the egg hatched rate of 70%by paraffin waxed,Egg sealing film sealed,shelling rate is 60%.These shelling are lower than those in the control group100%.The different opening positions.The hatching rate of chicken embryo is different.The egg was opened at the blunt end.The hatching rate of chicken embryo is 70%.the egg was opened at the blunt end,The hatching rate of eggs was 50%.These hatching rate of eggs are lower than those of control group.The hatching rate of the control group was 100%.We explore effects of different injecting instruments about hatching rate of chicken embryo.1mL syringe injected eggs,hatching rate was 60%.20μL micro-syringe injected eggs,hatching rate was 60%.40um diameter micro-needle injected of eggs,hatching rate of 70%.The hatching rate was lower than the control group100%.We explored effects of injected different solutions chicken embryo development.Trypan blue injected eggs,hatching rate was 0%.We injected eggs with PBS,the hatching rate was 25%.Physiological saline injected eggs,hatching rate was 30%.The hatching rate was lower than the control group 100%.Prodution of transgenic chicken by lentivirus:effects of blunt end uinjection of lentivirus with different titer on chick embryo development.We use lentivirus titer 108TU/mL injected eggs.The hatching rate was 50%.We use Lentivirus titer of 107 TU/mL injected of eggs,hatching rate was 50%.Lentivirus titer of 106 TU/mL injected of eggs,hatching rate of 65%.Lentivirus titer of 105 TU/mL injected of eggs,hatching rate of 65%.The incubation rate of saline group was 70%.The eggs were injected with 109 TU/mL lentivirus on the equator,the hatching rate was about 50%,and the shell number was about 10.Test of transgenic chicken embryos and chickens:we make PCR detected of blood and tissue DNA.Producing transgenic chicken was a useful method byblunt end injected.We use this method for prouducing tiangenic chickenWe tested the expression of target genes in chicken beak,pancreas,brain,muscle,stomach,lung,heart,bursa of Fabricius and kidney.This study confirmed that the equatorial microinjection can prouduce G0 LTF positive transgenic chicken.This help transgenic chicken production in the future and provide reference.Lentivirus mediated production of chicken oviduct will has a good foundation of bioreactor.