Insertion of the βGeo Promoter Trap into the Fem1c Gene of ROSA3 Mice

Abstract

ROSA3 mice were developed by retroviral insertion of the ␤Geo gene trap vector. Adult ROSA3 mice exhibit widespread expression of the trap gene in epithelial cells found in most organs. In the central nervous system the highest expression of ␤Geo is found in CA1 pyramidal cells of the hippocampus, Purkinje cells of the cerebellum, and ganglion cells of the retina. Characterization of the genomic insertion site for ␤Geo in ROSA3 mice shows that the trap vector is located in the first intron of Fem1c, a gene homologous to the sex-determining gene fem-1 of Caenorhabditis elegans. Transcription of the Rosa3 allele (R3) yields a spliced message that includes the first exon of Fem1c and the ␤Geo coding region. Although normal processing of the Fem1c transcript is disrupted in homozygous Rosa3 (Fem1c R3/R3 ) mice, some tissues show low levels of a partially processed transcript containing exons 2 and 3. Since the entire coding region of Fem1c is located in these two exons, Fem1c R3/R3 mice may still be able to express a putative FEM1C protein. To this extent, Fem1c R3/R3 mice show no adverse effects in their sexual development or fertility or in the attenuation of neuronal cell death, another function that has been attributed to both fem-1 and a second mouse homolog, Fem1b. Examination of ␤Geo expression in ganglion cells after exposure to damaging stimuli indicates that protein levels are rapidly depleted prior to cell death, making the ␤Geo reporter gene a potentially useful marker to study early molecular events in damaged neurons.Promoter trap experiments using the reverse orientation splice acceptor (ROSA) ␤Geo retroviral gene trap vector (9) have been useful in identifying genes involved in mouse development (4, 5). The coding region for the trap vector contains bacterial ␤-galactosidase fused in frame to neomycin phosphotransferase (Neo) to produce a fusion protein (␤Geo) that processes substrates like X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) and neomycin. Transcription of ␤Geo is dependent on the trap vector being inserted into the vicinity of an existing gene in the mouse genome, where the presence of a 5Ј splice acceptor facilitates the splicing of the ␤Geo coding region into the nearest upstream exon. The ROSA3 line of mice was initially part of 31 lines established using retroviral insertion of the ␤Geo vector into embryonic stem cells (9). The insertion of the ␤Geo gene caused embryonic recessive lethality in some of the lines. ROSA3 mice showed no overt defects, however, and exhibited a widespread pattern of staining by embryonic day 12.Here, we report the further characterization of the ROSA3 line of mice. As with the results seen with the embryos, ROSA3 mouse adult tissues show widespread staining (including selective staining of certain populations of neurons in the central nervous system) in ␤-galactosidase assays. Analysis of the genomic insertion site shows that the ␤Geo gene has been inserted into the first intron of Fem1c, the third member of a murine gene family that shares homolo...

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“…For these experiments, we used Fem1c Rosa3 (R3) mice, which contain the βGeo promoter trap reporter in the first intron of the Fem1c gene. Previously, we showed that mice express βGEO in an RGC-specific manner [12]. Additionally, we have observed a 75% decrease in βGEO total protein and enzyme activity [12], and a 50% decrease in Fem1c transcript levels, by 5 days post ONC (Figure 8A, B).…”

“…Previously, we showed that mice express βGEO in an RGC-specific manner [12]. Additionally, we have observed a 75% decrease in βGEO total protein and enzyme activity [12], and a 50% decrease in Fem1c transcript levels, by 5 days post ONC (Figure 8A, B). Thus, the R3 reporter allows for the rapid detection and quantification of changes in RGC gene expression.…”

Hydrocortisone Stimulates Neurite Outgrowth from Mouse Retinal Explants by Modulating Macroglial Activity

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“…Humans have a ubiquitously expressed 6.2-and 3.8-kb mRNA and a testis-specific 2.8-kb transcript. Schlamp et al (2004) reported a slight non-significant increase in the number of female pups in a mouse line with reduced expression of Fem1c, caused by insertion of the βGeo gene trap vector in the first intron of Fem1c gene, and concluded that the gene had no known effect on sexual development or fertility.…”