Abstract
Abstract: :
Purpose: The rod cGMP–gated cation channel ß–subunit is encoded by 33 exons within the Cngb1 locus. Several related alternatively spliced products are also generated from this locus in the retina and other tissues. To determine the function of the rod encoded proteins we created a Cngb1 targeted allele deleted for a predicted rod–specific promoter and the first two exons common to rod Cngb1 transcripts. Methods: The targeting construct consisted of the PGK promoter neomycin (Neo) expression cassette and 1.4 kb 5’ and 8 kb 3’ flanking region from Cngb1, creating a deleted region of 3.5 kb that includes exons 1 and 2, and a predicted promoter region. Genotypes were identified by PCR with Neo or Cngb1 specific primers. Northern analysis was done with a 5’ region probe common to all three retinal gene transcripts. Primer pairs spanning the Cngb1 coding regions and within the PGK–Neo cassette were used for RT–PCR. Results: Because non–retinal tissues, including testis, heart, brain, and kidney also express Cngb1 variants we did a gross examination of the mice. Externally the knockout mice (KO) are normal in size and appearance, and there are no apparent developmental or systemic abnormalities. PCR products of 1.6 and 1.75 kb were generated with Neo or Cngb1 specific products, respectively that differentiate the genotypes. RT–PCR revealed transcription of a Neo–Cngb1 hybrid mRNA missing exons 1 and 2. Exons 3–31 of Cngb1 and exon 12a specific to GARP–2 could be amplified from KO cDNA. Sequence analysis of the hybrid transcript revealed an intact Neo coding sequence and stop codon. Northern analysis of total RNA from each genotype identified prominent 1.5 and 6.0 kb transcripts in +/+ and +/– mice that were not detected in the homozygous KO. The entire intron/exon structure of murine Cngb1 was determined by partial sequence analysis and comparison of mouse and human genome and cDNA sequences. This analysis revealed in GARP–1 a new exon, 16a that is also utilized in rat olfatory ß–subunit, and an extension of exon 18. Conclusions: The Cngb1 KO appears to be a true null that produces a detectable but low abundance Neo hybrid transcript that is not capable of generating Cngb1 related protein products. The alternate exon identified for GARP–1 may be functionally important because it is also found in the olfactory CNG ß–subunit.
Keywords: transgenics/knock–outs • retina • transcription