Figure 2

Generation of B6.Cg-Tg(Kcnj10-ZsGreen)^skMH (Kcnj10-ZsGreen) fluorescent transgenic mice. (a) A schematic of the BAC clone RP23-157J4 expected to contain mouse genomic DNA (gDNA) including promoter, coding sequence and regulatory elements responsible for the endogenous expression of Kcnj10, is shown on the top of the panel. The BAC backbone contains a gene of resistance to chloramphenicol (cpl^r). The BAC and a synthetic donor DNA containing the coding sequence of ZsGreen followed by bgh-polyA(pA) sequences, and a gene conferring kanamycin/neomycin resistance (kan^r/neo^r) surrounded by FRT sites, were combined in recombineering competent bacteria. The insertion of this synthetic donor in place of the coding sequence of Kcnj10 by homologous recombination was made possible by the insertion in 5′ and 3′ of this cassette of 80 bp fragments of DNA homologous to the target regions in the BAC (darker blue regions). Introduction of the cassette into the BAC resulted in kan^r of the bacteria. DNA from kan^r BACs were further analyzed to identify correctly modified BACs. The kan^r cassette has both prokaryotic and eukaryotic (mouse Pgk 1) promoters. Black lines indicate regions of recombination between mouse gDNA in the BAC DNA and homologous sequences introduced by PCR in the synthetic DNA donor plasmid. The kan^r/neo^r cassette was removed by expression of flipase (FLP) recombinase. DNA from kanamycin sensitive BACs were analyzed to identify correctly modified BACs. A final recombination step replaced the BAC-backbone internal loxP site with ampicillin resistance (step not shown). (b) Mice carrying Kcnj10-ZsGreen transgene were identified by PCR. F6/R6 primer pair targets the DNA region between the promotor of Kcnj10 and the 5′ sequence of ZsGreen. F1/R1 primer pair targets the 3′ sequence of ZsGreen and its polyA region. Amplicons of 156 bp with primers F6/R6 and 924 bp with primers F1/R1 indicate the presence of the transgene. These amplicons were detected in Kcnj10-ZsGreen mice gDNA but not in the gDNA of their wildtype littermates. The sequence of these primers is reported in Table S11. H₂O: negative control without gDNA template. DNA size marker (ThermoScientific GeneRuler 100 bp Plus DNA ladder SM0323, Waltham, MA) was used. (c) To evaluate Kcnj10-ZsGreen transgene copy number, ddPCR was performed on the gDNA of ZsGreen hemizygote mice from the founder lines 850 and 858 and their wildtype (WT) littermates, after digestion with MseI. The gene Rpp30, located on chromosome 19 in mice, was used as a reference. ddPCR revealed the presence of ZsGreen targeted amplification in as many droplets as Rpp30 positive droplets, supporting the fact that hemizygous 850 and 858 ZsGreen mice each carry two copies of the ZsGreen transgene.