Strictly Regulatory Expression Of HCV NS3/4A Protein In Transgenic Mice

The hepatitis C virus (HCV) is the main pathogen causing chronic liverdiseases，and approximately3%of the world population has infected withHCV．Because of highly genetic variability of HCV，up to now，there have beenno effcient vaccines and specific therapies for prevention and treatment againstHCV infection．It has become a global public health problem．Since HCV was first isolated and identified in1989，the progress in studyof prevention and treatment against HCV infection has moved forwardslowly．This is mainly due to the strict species tropism of HCV infection whichlead to lack of effective，stable experimental animal models for study．This notonly seriously impedes the study of HCV infection and pathologic mechanism，but also greatly limits the development in antiviral drugs，as well as inpreventive and therapeutic vaccines．The viral genome encoded NS3/4A protein is a multifuctional protein posessing serine protease activity and helicase activity．In vitro cell culturebased studies indicate that the NS3/4A protein plays crucial roles in the proteinprocess of viral RNA translated precursor and in the process of RNA replicationand virus assembly．Thus，NS3/4A protein has become one of the mostsignificant targets of direct antiviral agent(DAA) development．Moreover，theinteraction between host proteins not directly participating in the process ofHCV replication and NS3/4A protein closely related to the virus-mediatedimmune evasion and persistence of infection．Therefore，it seems that inhibitorsdirectly targeting NS3/4A protein have a dual role in HCV infected cells，namely blocking the virus replication cycle and restoring the antiviral innateimmune response．The study here aimed to establish a transgenic mouse model stably andconditionally expressing NS3/4A protein via combination of Tet-On regulatorysystem and Cre/LoxP gene-knockout system， which may make a solidfoundation for further study of the interaction between NS3/4A protein and thehost proteins，as well as for screening and evaluation of NS3/4A proteaseinhibitor．1. Construction of an recombiant vector expressing NS3/4A proteinunder the dual control of Tet-On regulatory system and Cre/LoxPgene-knockout system.To construct a recombinant vector expressing NS3/4A protein under thedual control of Tet-On regulatory system and Cre/LoxP gene-knockout systemby using DNA cloning technology，the sequences of LoxP、Fluc、BGHPolyA、LoxP and NS3/4A were orderly inserted into the multiple cloning site in thedownstreem of tet-responsive promoter．The vector，pBI-G//LoxP-FLuc-BGHPolyA-LoxP-NS3/4A，was co-transfected with pTet-On and pBI-G//Cre intoCHO cells and the transfected cells were induced with Dox．Protein expression detection demonstrated that only the co-existence of Tet-On system andCre/LoxP system resulted in expression of NS3/4A protein by the recombinantvector．Besides，the appropriate introduce of reporter Fluc into the recombinantvector，would greatly facilitate latter studies by using the real-time and sensitivein vivo bioluminescent imaging technology．2. Establishment of NS3/4A transgenic mouse lines and screening ofNS3/4A/Lap double transgenic mice efficiently expressing reporter Fluc.NS3/4A transgenic vector was obtained by restriction enzyme linearizationof the vector pBI--G//LoxP-FLuc-BGH PolyA-LoxP-NS3/4A and zygotemicroinjection produced6NS3/4A transgenic founder mice which wereidentified by PCR and Southern Blot．These transgenic founder mice werecrossed with the established homozygotic Lap transgenic mice liver-specificallyand constitutively expressing reverse tet-controlled transcriptional activator(rtTA)．All of the F1mice were first genetyped by PCR of the transgenicfragments using genomic DNA as templates．Subsequently，both of NS3/4A andLap transgenic positive mice were phenotyped by in vivo bioluminescentimaging(BLI) to detect the expression of Fluc reporter after persistentlyinducing with Dox．These bioluminescent-signal positive NS3/4A/Lap doubletransgenic mice (F1) were crossed with homozygotic Lap transgenic mice andoffsprings(F2) displaying complete consistence between genetype andphenotype were screened by PCR and BLI．Similarly，each generation ofNS3/4A/Lap double transgenic mice were crossed with homozygotic Laptransgenic mice and offsprings were screened by PCR and BLI to establishNS3/4A transgenic mouse lines，untill each of NS3/4A/Lap double transgenicoffsprings from the same parernts had a completely identical phenotype(identical characteristic of transgene expression) with others．Western Blot ofthe tissues obtained from these NS3/4A/Lap double transgenic mice showed that the reporter Fluc were conditionally expressed in the liver of Dox-inducedNS3/4A/Lap double transgenic mice．3. Screening of Dox inducible NS3/4A/Lap/LC-1triple transgenic miceefficiently and conditionally expressing NS3/4A proteinThe identifid NS3/4A/Lap double transgenic mice were mated withLap/LC-1double transgenic mice which liver-specifically and conditionallyexpress Cre recombinase under control of Tet-On system．Offsprings werescreened by PCR for genetype identification and BLI for phenotypeconfirmation to obtain NS3/4A/Lap/LC-1triple transgenic mice．Western Blotand Immunohistochemisty staining of the tissues obtained from theseDox-induced NS3/4A/Lap/LC-1triple transgenic mice demonstrated that theNS3/4A protein and Cre recombinase were conditionally expressed in the liverof triple transgenic mice．The different expression pattern of NS3/4A/Lap/LC-1triple transgenic mice from that of NS3/4A/Lap double transgenics confirmedthat NS3/4A protein was only expressed under the dual control of Tet-On systemand Cre/LoxP system with characteristic of controllability and specificity．Above all，this study successfully established a transgenic mouse model inwhich the NS3/4A protein were conditionally expressed in liver under the dualcontrol of Tet-On system and Cre/LoxP system．Therefore，this transgenicmouse model provide a useful tool for further study of the interaction betweenNS3/4A protein and the host in vivo，as well as for inhibitor screening andevaluation．...