The ROSAβgeo26 (ROSA26) mouse strain was created through random retroviral gene trapping in embryonic stem cells. This strain exhibits widespread expression of the βgeo reporter gene, which is used as a marker for chimera and transplantation studies. The gene trap vector has integrated into a region that produces three transcripts. Two of these transcripts, which are absent in homozygous ROSA26 mice, originate from a common promoter and share identical 5' ends but do not contain significant open reading frames (ORFs). The third transcript, originating from the reverse strand, shares antisense sequences with one of the noncoding transcripts and potentially encodes a novel protein of at least 505 amino acids that is conserved in humans and Caenorhabditis elegans. Gene traps are a general strategy to identify genes with discrete expression patterns during development and differentiation. The trap vectors contain a reporter gene, typically β-galactosidase (β-gal), which is not expressed unless it integrates into an intron or exon of a transcription unit. The integration results in reporter gene expression that reflects the expression pattern of the endogenous gene or is influenced by nearby transcriptional regulatory elements. The reporter gene also provides a molecular tag for cloning the trapped gene. These techniques have been used to identify novel genes with diverse reporter gene expression patterns, providing valuable tools for studying normal development and understanding the developmental consequences of specific mutations. The ROSA26 strain displays ubiquitous expression of the reporter gene during embryonic development and has been useful as a marker line in chimera experiments. In this study, the researchers extended these observations by demonstrating ubiquitous β-gal activity in various tissues and hematopoietic cells and by illustrating the use of this strain for bone marrow transfers. They characterized the region in which the reporter gene had integrated and showed that both DNA strands of the ROSA26 genomic region are transcribed, producing convergent and antisense RNAs. The ROSA26 region produces three transcripts. 5' RACE was used to identify exons from the trapped gene. The RACE product contained 130 bp of unique sequence. This sequence was used as a probe on Southern blots of StuI-digested DNA from wild-type, heterozygous, and homozygous ROSA26 mice. The probe identified a restriction fragment length polymorphism (wild-type band of ≈7 kb and mutant band of ≈12 kb) that cosegregates with the ROSA26 allele. The unique sequence was used to design primers for 3' RACE. Two classes of products were obtained. Both contained identical 5' ends but diverged at their 3' ends. These 3' RACE products were used as probes on cDNA libraries. Multiple cDNAs were obtained for one of the two RACE products and their sequence was used to pieceThe ROSAβgeo26 (ROSA26) mouse strain was created through random retroviral gene trapping in embryonic stem cells. This strain exhibits widespread expression of the βgeo reporter gene, which is used as a marker for chimera and transplantation studies. The gene trap vector has integrated into a region that produces three transcripts. Two of these transcripts, which are absent in homozygous ROSA26 mice, originate from a common promoter and share identical 5' ends but do not contain significant open reading frames (ORFs). The third transcript, originating from the reverse strand, shares antisense sequences with one of the noncoding transcripts and potentially encodes a novel protein of at least 505 amino acids that is conserved in humans and Caenorhabditis elegans. Gene traps are a general strategy to identify genes with discrete expression patterns during development and differentiation. The trap vectors contain a reporter gene, typically β-galactosidase (β-gal), which is not expressed unless it integrates into an intron or exon of a transcription unit. The integration results in reporter gene expression that reflects the expression pattern of the endogenous gene or is influenced by nearby transcriptional regulatory elements. The reporter gene also provides a molecular tag for cloning the trapped gene. These techniques have been used to identify novel genes with diverse reporter gene expression patterns, providing valuable tools for studying normal development and understanding the developmental consequences of specific mutations. The ROSA26 strain displays ubiquitous expression of the reporter gene during embryonic development and has been useful as a marker line in chimera experiments. In this study, the researchers extended these observations by demonstrating ubiquitous β-gal activity in various tissues and hematopoietic cells and by illustrating the use of this strain for bone marrow transfers. They characterized the region in which the reporter gene had integrated and showed that both DNA strands of the ROSA26 genomic region are transcribed, producing convergent and antisense RNAs. The ROSA26 region produces three transcripts. 5' RACE was used to identify exons from the trapped gene. The RACE product contained 130 bp of unique sequence. This sequence was used as a probe on Southern blots of StuI-digested DNA from wild-type, heterozygous, and homozygous ROSA26 mice. The probe identified a restriction fragment length polymorphism (wild-type band of ≈7 kb and mutant band of ≈12 kb) that cosegregates with the ROSA26 allele. The unique sequence was used to design primers for 3' RACE. Two classes of products were obtained. Both contained identical 5' ends but diverged at their 3' ends. These 3' RACE products were used as probes on cDNA libraries. Multiple cDNAs were obtained for one of the two RACE products and their sequence was used to piece