Mouse Retina-specific α-Cre Transgene:Identification Of Its Integration Site And Its Expression In Olfactory Neurons

Objective:In α-Cre mouse line,Cre recombinase is expressed specifically in retina.This mouse line is generated by pronuclear microinjection of α-Cre transgene plasmid DNA into germline cell,thus the transgene inserted into mouse genome randomly.Without knowledge of the integration site,two consequences may come with it.One is the differentiation between hetero-and homozygous animals.If the integration disrupts an endogenous gene,homozygous α-Cre may potentially interfere with interpretation of the transgenic mouse phenotype.Another problem is the planning of breeding crosses,especially between animals carrying a conditional allele and a given Cre allele in case these alleles are on the same chromosome.In this study,we examined and characterized the expression pattern of Cre recombinase in the retina of α-Cre mouse line.Then we employed targeted locus amplification(TLA)to precisely map the α-Cre transgene integration site on mouse genome.Materials and Methods:This study is composed of 7 parts.First,α-Cre mice were crossed with Cre reporter lines(R26R and Ai14 mice).The heads and retinas of these mice at embryonic day 12(E12),E14,E16,at birth(P0),postnatal day 8(P8)and P18 were collected and subjected to X-Gal and immunofluorescence staining to observe the transgene expression pattern in the retina of α-Cre mouse.Second,we crossed Rbf/f;Bax+/-;α-Cre males with Rbf/f;Bax-/-females.Genotypes of their offspring and recombination rate were analyzed to investigate the genetic linkage between α-Cre and Bax gene.Third,we employed TLA to map the α-Cre transgene integration site on mouse genome as well as its internal sequences.Fourth,following the validation of TLA results with polymerase chain reaction(PCR)and Sanger sequencing,we designed multiple allele-specific genotyping assays to differentiate hetero-and homozygous α-Cre transgene.Fifth,integration site of α-Cre transgene on chromosome 7 was analyzed using online UCSC genome browser.It was integrated among a series of olfactory receptor gene clusters.We harvested heads of R26R;α-Cre and Ai14;α-Cre mice at E14,E16 and P0.X-Gal and immunofluorescence staining were performed to examine transgene expression pattern in mouse olfactory system.Sixth,in order to test the feasibility of applying α-Cre mice to study olfactory system,we used buried food pallet(BFP)test to examine the olfactory function of Rbf/f;α-Cre mice and control mice(α-Cre or No Cre mice)at P30.Seventh,we harvested the retina and nasal mucosa of Rbf/f;α-Cre mice and control mice(α-Cre or No Cre mice)at P13.Quantitative Real-time PCR(q RT-PCR)was then performed to analyze the expression level of olfactory receptor genes flanking α-Cre transgene integration site.Results:1.Temporally,α-Cre expression in the retinal progenitor cells started from E10 and was silenced gradually after E12.From E16,it started to express in amacrine cell which had exited the cell cycle.Spatially,in retinal progenitor cells,α-Cre transgene mainly expressed in the nasal and temporal peripheral retina with distalhigh to proximallow pattern.In amacrine cells,α-Cre expression was found across whole retina with no apparent gradient.2.We bred Rbf/f;Bax+/-;α-Cre males with Rbf/f;Bax-/-females and obtained 96 pups.Only 5 pups with the genotype of Bax-/-;α-Cre,suggesting α-Cre and Bax-alleles may be on the same chromosome(chromosome 7)with at least 20 Mb genetic distance(recombination rate of 5.2%).3.According to TLA results,α-Cre transgene has integrated in mouse chr7: 92408005-92435609bp)with a 27 kb deletion.4.According to PCR and Sanger sequencing validation,all sequences were consistent with TLA results except that there was no SV40 poly(A)sequence at the breakpoint of β-globin intron.At the same time,sequences of five linkers inside α-Cre transgene were determined for the first time.Based on detailed information of transgene integration site,allele-specific genotyping assays were designed and tested.Multiple sets were proven capable of efficient differentiation between hetero-and homozygous α-Cre transgene.5.With online UCSC genome browser,we found α-Cre transgene was integrated among a series of olfactory system-related genes.Upstream α-Cre transgene were two Vmn2 r clusters(Vmn2r69-73,Vmn2r77-79,)and one Olfr cluster(Olfr292-310).Downstream included Vmn2r65-68 and Olfr290/291 genes.Vmn1r55-185,Vmn2r28-56,Vmn2r57-63 and Omp(olfactory marker protein)were located on chr7 as well.α-Cre expression in olfactory system included main olfactory sensory epithelium(MOE),vomeronasal organ(VNO)and their axons projecting to olfactory bulb or accessory olfactory bulb.As in retina,it was gradually silenced during development.6.Olfactory function test of Rbf/f;α-Cre mice: 27 mice were included in BFP test and divided into three groups,Rb-Cre,Cre and No Cre group.BFP score(unit: 1/second×1000): 63.59±21.51 for Rb-Cre group,58.12±19.42 for Cre group and 45.45±30.16 for No Cre group.There was no significant difference of BFP score among three groups(P=0.282).7.Transcription level of olfactory receptor genes in retina and nasal mucosa: Vmn2r67,Vmn2r68,Vmn2r69,Vmn2r70,Olfr291,Olfr310,Omp were chosen for q RT-PCR analysis.No significant differences were found between No Cre,Cre,and Cre-Rb groups of the nasal mucosa and retina of P13 mice.Conclusion:In this study,we investigated the expression pattern of α-Cre transgene in mouse retinal progenitors and amacrine cells.α-Cre mouse line was believed to express Cre recombinase specifically in retina without detailed knowledge of its integration site.By breeding with Bax KO mice,we found the α-Cre transgene was linked to Bax gene,which located in Chromosome 7.Further we mapped its precise integration site on Chromosome 7 and all sequences information of α-Cre transgene by TLA technology.As the integration site is among a cluster of olfactory genes,α-Cre also expresses in the murine olfactory system,indicating the regulation elements of olfactory receptor genes can affect the expression of α-Cre transgene.However,we didn’t detect any effects of α-Cre transgene(including the enhancer and promoter of Pax6 gene)on the expression of flanking olfactory genes in the nasal mucosa and retina.Our preliminary results indicated that knocking out Rb gene in the olfactory neurons using α-Cre has no major effects on olfactory function at P30,future study was needed to reveal its mechanism.Together,this study provided detailed knowledge of α-Cre transgene integration site and internal sequences,allowing for avoiding futile planning of crosses,improving genotyping assays and interpreting phenotypes obtained for future researchers.