Figure 2

Construction and characterization of the Umodl1 BAC transgene. (a) The size and position of RP24-430m9 Umodl1 BAC are indicated. Linearized targeting vector, pW223, was used to modify the BAC. Mouse Umodl1 is comprised of 22–24 exons that spread over 56 kb in length on chromosome 17, and produces four alternative splicing transcripts. The BAC clone RP24-430m9 contains a 176 kb genomic fragment that harbors the entire Glp1r and Umodl1, as well as the first three exons of Abcg1at the 3′ end. This BAC clone was modified by inserting the reporter ires.lacZ.GT1.2/neo into the 3′-UTR of Umodl1. A unique NotI restriction site located in the 4th intron of Glp1r was utilized to inactivate Glp1r. From the resultant pW224, functional Umodl1, β-galactosidase and neomycin phosphotransferase can be produced. Tg mice were generated by pronuclear injection of NotI-digested pW224. (b) Stable pW224 Tg lines were verified by genomic PCR using Oligo 251 and Oligo 514. (c) Copy number of each Tg line was estimated by the Taqman real-time PCR method. (d) Quantitative PCR to compare Umodl1 mRNA expression in the olfactory bulbs of different Tg lines to the WT control. GAPDH mRNA was amplified in parallel and used as the quantity control. Data were obtained from three independent repeats. *P<0.05