Generation Of Double Reporter Gene Fluc-mRFP Transgenic Mice By Lentivirus-mediated Gene Transfer

Objectives:1) Master technique for generating transgenic mice by lentivirus-mediated delivery of foreign gene(s) into zygotes2) Generate the double reporter gene Fluc-mRFP trausgenie mice,which will be potentially employed to provide the phenotype-defined double reporter gene-labelled cells for transplantation,followed by in vivo monitoring the fates of the transplented cells and their progeny in a sensitive,real-time,noninvasive and visualizational mannerMethods:1) Lentiviral vector of phUb-Flue-mRFP identified by enzyme digestion and PCR(1) Identification of phUb-FR by enzyme digestion:phUb-FR was cut by Notâ… ,Mluâ… and BamHâ… ;(2) Identification of phUb-FR by PCR:primers P1/P2,P3/P4 and P5/P6(specific for Fluc,mRFP and ttk,respectively) were employed to amplify Fluc,mRFP and ttk (from phUb-FR as template),respectively.2) Production and identification of lentivirus carrying hUb-FR geneAccording to the standard protocol from Invitrogen,lentivirus carrying hUb-FR gene was produced,followed by confirming that lentivirus harboring hUb-FR gene was successfully made.3) In vitro assay for the expression of Flue and mRFP genes in the cell levelPrior to generate FR transgenic mice,293FT cells were infected with the lentiviruses harboring FR gene to further verify whether the double reporter genes Fluc and mRFP were normally expressed in cell-base model by screening for +/- mRFP expression under fluorescence microscopy and by detecting the expression of Fluc and mRFP genes via RT-PCR.4) Generation of Fluc-mRFP transgenic mice by lentivirus-mediated gene transferLentiviruses harboring double reporter gene Fluc-mRFP(FR) were subzonally injected into single-cell fertilized eggs of ICR mice to infect fertilized eggs,and subsequently embryos were transplanted into the pseudopregnant mice to attain F0 mice,followed by screening Fluc-mRFP transgenic mice(Founder,F0) by mRFP assay under fluorescent stereo microscope and Fluc assay via in vivo bioluminescence imaging,and subsequently confirm that FR gene actually integrated into the genome of mRFP-positive FR transgenic mice by PCR-based genotyping.5) Mouse propagation and transgene transmissionAt 6-8 wk of age,founder shown to be transgenic for the Fluc-mRFP was mated with wildtype ICR mouse to produce F1 generation,followed by mRFP assay under fluorescent stereo microscope to demonstrate whether Fluc-mRFP transgene in Fluc-mRFP transgenic mice(F0) could be transmitted to the next generation(F1).6) Detection of mRFP expression in adult organs of FR transgenie mice(F1) under fluorescent stereo microscopeResults:1) phUb-Fluc-mRFP identified by enzyme digestion and PCRLentiviral vector of phUb-Fluc-mRFP(used to produce lentivirus,followed by generating the transgenic mice by lentivirus-mediated gene transfer) was confirmed to be right by enzyme digestion and PCR.2) Lentivirus production and in vitro assay for Fluc and mRFP expression By mRFP assay under fluorescence microscopy,293FT cells were confirmed to be efficiently infected by concentrated lentivirus.Furthermore,prior to generate FR transgenic mice,cell-based model was used to demonstrate that the double reporter genes Fluc and mRFP were normally expressed in 293FT cells infected with the lentiviruses harboring FR gene. 3) Generation of Fluc-mRFP transgenic mice by lentivirus-mediated gene transferTransgenic mice were generated by subzonal injection of lentivirus harboring Fluc-mRFP gene into single-cell fertilized eggs of ICR mice to infect fertilized eggs. 525 virus-injected eggs were transplanted into 28 pseudopregnant mice to attain 136 F0 mice(potential transgenic founders).Founder mice were selected from 136 potential transgenic founders(F0) by mRFP assay under fluorescent stereo microscope,and subsequently confirm the results of mRFP assay by PCR for genotyping.A total of 63 PCR-positive founders was attained,all of which transmitted Fluc-mRFP transgene to their offspring.Ratio of founders to born mice was 46%.47 out of 63 PCR-positive founders normally expressed mRFP gene, whereas 16 out of 63 showed almost no fluorescent signal.Among these 47 founders expressing mRFP gene,16 PCR-positive founders illustrated strong red fluorescence, while 31 showed relatively weaker signals when observed under fluorescent stereo microscope.In addition,Fluc expression in mRFP-positive Fluc-mRFP transgenic mice could be tested through in vivo bioluminescence imaging.4) Transmissibility of the foreign transgeneF1 progeny inherit and express Fluc-mRFP transgene from Fluc-mRFP transgenic founder(F0).5) mRFP expression in adult organs of FR transgenic mice(F1)Red fluorescence was detected in tissue samples including brain,intestine,kidney, spleen,skin,hair,and etc isolated from the FR transgene-positive mice but not in wildtype mice and control littermates.Conclusions:1) Master technique for rapidly generating transgenic mice by lentivirus-mediated gene transfer2) Efficiently and rapidly produce Fluc-mRFP transgenic mice3) Fluc-mRFP transgene could be transmitted from founders to subsequent generation4) Normal expression of mRFP transgene in multi-organs of Fluc-mRFP transgenic mice(F0 and F1)5) Fluc expression in FR transgenic mouse could be detected by bioluminescence imaging...