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. 2018 Apr 26;43(5):367–378. doi: 10.1093/chemse/bjy024

Figure 1.

Figure 1.

Genotype and gene expression validation of successful excision of Rgs21 exon 5. (A) Architecture of Rgs21 locus on chromosome 1 of C57BL/6J mice, as obtained from GenBank record NC_000067.6 and spanning its 5 exons (Ex1–Ex5) as denoted by its RNA transcript (NM_001290269, purple) and its encoded RGS21 polypeptide (NP_001277198.1, pink). (B) Illustration of wild-type Rgs21 surrounding exon 5, the predicted insertion of loxP recombination sites and neomycin (“neo”) selection cassette by homologous recombination, and the predicted result of Flp recombinase excision. Forward (cyan) and reverse (orange, red) primers used in detecting Cre-mediated recombination of loxP sites are indicated as arrowheads. (C) Southern blot validation of successful Flp recombinase excision of neo cassette, based on AflII digestion of genomic DNA from indicated mice (or C57BL/6 control mouse, “WT”), subsequent blotting on nylon membrane, and hybridization with an external 3′ DNA probe (indicated in B). (D) Ear-punch DNA samples from 4 indicated progeny of an Rgs21Δ5/+ × Rgs21Δ5/+ mating were genotyped by PCR. Specific primers (denoted in B) were designed around the floxed exon 5 portion of Rgs21 to discriminate between unexcised (Rgs21+/+) and Cre recombinase-excised (Rgs21Δ5/Δ5) genomic DNA. (E) Data from qRT-PCR (SYBR Green detection) of the Rgs21 mRNA transcript, which is seen to be completely absent in Rgs21Δ5/Δ5 mice and absent in non-gustatory (“NG”) epithelial tissue from Rgs21+/+ mice, but detectable in tongue and enriched in circumvallate papillae (“CV”) tissue from Rgs21+/+ mice.