Fig 1. Generation of Rm155LG transgenic mice.

(A) Schematic diagram of the Rm155LG transgenic construct used to generate Rm155LG transgenic mice. A potent, ubiquitous CMV/β-actin promoter in the vector pRm155LG was used to drive a series of cassettes, including a floxed mRFP followed by a triple transcription-stopping polyA sequence (3×PolyA) and a downstream internal ribosome entry site (IRES)-based bicistronic transcript, including open-reading frames of mouse miR-155 and a multifunctional marker consisting of firefly Luc fused to eGFP with a transmembrane-localizing domain (Luc-TMeGFP). The primer pair P1/P2 represented by small arrows were used in PCR analysis of genotype to detect reporter transgene mRFP. Only mRFP will be transcribed and expressed properly from this construct, while Cre-mediated recombination occurs, the floxed mRFP+3×PolyA is excised, and the downstream, bicistronic transcript is activated. The multifunctional marker will be expressed, replacing mRFP in Cre-activated cells. The construct map is not drawn to the scale. Abbreviations: CAG promoter: CMV early enhancer/chicken β actin promoter; mRFP: monomeric red fluorescent protein; Luc: firefly luciferase; EGFP: enhanced green fluorescent protein; pA: polyadenylation signal; The black triangle: lox P site. (B) Screening Rm155LG transgenic founders by in vivo non-invasive fluorescence imaging. Three foster mothers gave birth to three, two and three F0 pups, respectively; three mRFP-positive Rm155LG transgenic mice (referred to as 1107^#, 1108^# and 2458^#) with strong red fluorescence were found via mRFP assay by using the Xenogen IVIS Lumina Imaging System 2–3 days after birth. (C) F1 progeny inherit and express mRFP transgene from three founders. Offspring shown in Fig. 1C-a,b,c were derived from the mating between founder 1107^#, 1108^# or 2458^# and wildtype mouse, respectively. A fraction of founder offspring with mRFP fluorescence showed that all of three founders could transmit Rm155LG transgene to subsequent generation (i.e., F1). (D) mRFP-positive founders verified for Rm155LG transgene presence by PCR analysis. Three mRFP-positive mice (i.e., 1107^#, 1108^# or 2458^#) and one mRFP-negative mice (i.e., 1109^#) were individually analyzed by PCR for the genomic integration of transgene with tail biopsy-derived DNA from mice (1107^#, 1108^#, 1109^# and 2458^#). PCR products were amplified by the primer pair P1/P2 (specific for mRFP) shown in Fig. 1A. lane PC: positive control (pRm155LG as template); lane NC: negative control using genomic DNA from WT mouse as template. Data are representative of three independent PCR experiments that yield similar results. (E) Rapidly and readily distinguishing homozygous from heterozygous Rm155LG transgenic alleles by in vivo fluorescence imaging.