Targeted expression of Cre recombinase to myelinating cells of the central nervous system in transgenic mice

We have generated transgenic mice which express Cre recombinase under the control of the mouse myelin basic protein (MBP) promoter. Previous studies have shown that 1.3 kb of 59 flanking sequence from the mouse MBP gene is sufficient to direct expression of heterologous genes specifically to myelinating oligodendrocytes in vivo (Turnley et al., 1991; Yoshioka et al., 1991). To generate transgenic mice, pronuclei of FVB/N fertilized eggs were injected with the MBPCre transgene (Fig. 1A). Two lines, MPBCre and MBPCre, that showed detectable amounts of Cre protein in brain by immunoblotting (data not shown) were chosen for further analysis. Mice of both Cre lines were fertile, transmitted the transgene in a Mendelian fashion, and showed no signs of disease. To test the specificity of Cre-mediated recombination, MBPCre-expressing transgenic mice were crossed to animals harboring the reporter transgene floxLacZ (Akagi et al., 1997). A modified PCR-based approach was used to detect the recombination event in the various tissues of MPBCre;floxLacZ mice. Recombination of the floxLacZ transgene was scored by amplification of a 384 bp PCR product using primers AG10 and Z6 (Fig. 1B). These primers also allowed amplification of a 1972 bp product, corresponding to the nonrecombined transgene, which was observed in all samples (Fig. 1B). In floxLacZ mice carrying the MBPCre –9 transgenes the 384 bp fragment specific for the deleted transgene was amplified principally in the CNS samples analyzed, thus confirming the specificity of this promoter for the CNS. However, possibly as the result of ectopic Cre expression, recombination was also observed in testis and pituitary gland of MBPCre;floxLacZ mice and in the heart of MBPCre; floxLacZ mice. Despite Cre expression in the testis, offspring from crosses of MBPCre;floxLacZ males to wild-type females inherited the nonrecombined floxLacZ transgene. Thus, MBPCre transgenic mice do not delete a floxed DNA fragment in the germline. Lower yields of the 384 bp fragments were amplified from tissues of MBPCre;floxLacZ mice compared to those in the corresponding MBPCre;floxLacZ tissue samples suggesting a lower rate of recombination. The difference in recombination efficiency of the two MBPCre transgenic lines was further documented by Southern blot analysis. CNS-specific deletion of the floxLacZ transgene was detected in mice carrying the MBPCre transgene (Fig. 1C), and the recombination frequency in brain (cortex) and cerebellum was estimated to be 44% and 50%, respectively. No recombination was detected by Southern blot analysis of CNS samples of MBPCre;floxLacZ mice indicating that the recombination frequency was relatively low as it was detected only by PCR analysis. To determine the localization of Cre-mediated recombination in the CNS, various samples from of MPBCre ;floxLacZ mice were analyzed for expression of b-galactosidase by X-Gal staining. In floxLacZ mice carrying the MBPCre transgene, specific staining was observed in various regions of the CNS. Blue-stained cells were found essentially in the white matter in brain (Fig. 2A) and spinal cord (Fig. 2B). No X-Gal conversion was detected in the spinal nerves (Fig. 2B), confirming that the 1.3-kb fragment of the MBP promoter sequence does not include elements that stimulate transcription in Schwann cells (Goujet-Zalc et al., 1993; Gow et al., 1992). Intense blue staining was also observed in the optic nerve (Fig. 2D). In MBPCre;floxLacZ mice scattered blue-stained cells were detected only in optic nerve and brain (Fig. 2C). Altogether, the expression profile of these MPBCre –9 transgenes is similar to that of MBPLacZ in transgenic mice generated using the same fragment of the MBP promoter (Gow et al., 1992). These MPBCre mice will be useful for the