Supplementary Figure 7: Generation and analysis of transgenic Brec1 mice.

(a) Schematic representation of the Brec1 expression cassette. Brec1 is expressed from the cytomegalovirus (CMV) immediate early enhancer-chicken beta-actin hybrid (CAG) promoter. Breeding strategy following generation of Brec1 founder mice by pronuclear injection: WT, wild type mouse; TGM, transgenic mouse. The number of offspring and the TG carriers indicated for the first and second generation show that the transgene is inherited with a Mendelian ratio. (b) Agarose gel showing PCR-based genotyping of one founder (lane 2) and three transgenic offspring (lanes 3-5) using Brec1-specific primers. WT, wild type control (lane 1); M, DNA marker lane. (c) Agarose gel showing PCR-based analysis of cDNA generated by reverse transcription of mRNA isolated from WT animal (control, lane 1) and three transgenic Brec1 mice (TGM1-TGM3, lanes 2-7): +, reverse transcriptase added to RT reaction; -, no-reverse transcriptase control. (d) Schematic representation of the lentiviral reporter construct. Important elements are indicated. The vector backbone has been previously described in detail²⁹ (e) Schematic drawing of the primer binding sites within the lentiviral reporter construct. Important elements are indicated and the size of the expected amplicon for primers P3 and P4 is shown. Brec1-mediated recombination leads to the formation of a circular excision product that can be detected employing primer P1 and P2. (f) Schematic of the 600 bp amplification product for primers P1 and P2. A sequencing read is shown confirming the expected sequence. The loxBTR sequence is marked. (g) Confirmation of lentiviral integration of the reporter construct. PCR assays for indicated animals is provided. The 1.7 kb fragment resulting from amplification with primers P3 and P4 is marked by an asterisk. (h) Confirmation of Brec-1 mediated recombination. Gel showing PCR analysis using DNA isolated from cells of indicated animals. The product showing the recombined band is indicated with P1/P2.