Fig. 4. Identification of transgene insertion site. (A) A Southern blot of genomic DNA from wild-type and transgenic mice, digested with the three restriction enzymes indicated and probed with a 3'-insertion site-specific probe. The arrows indicate the three single, novel bands hybridizing to the probe in the DNA from the transgenic mice, indicating the likelihood of a single transgene insertion site. (B) A PCR-based analysis of genomic DNA from one litter of interbred transgenic mice, indicating the PCR products that were specific for the presence of the transgene (Transgene-specific) and those that were specific for the endogenous sequence that was interrupted by the transgene (Insertion site-specific). The transgene specific primers were 5'-AGCCAGTAATAAGAACTGCAGA-3' and 5'-GGCACTCTTAGCAAACCTCAGG-3', which correspond to bp 264-285 of the human cytochrome P450 cDNA clone accession number NM_000775.2 and bp 5225-5246 of the mouse -myosin heavy chain promoter clone Accession Number MMU71441, respectively. The insertion site specific primers were 5'-CATGGAAAGGGCAGAGTGAGC-3' and 5'-GGCCATTGTCACCACTCGTAA-3', which correspond to bp 732-752 and bp 323-343 of mouse trace archive sequence gnl|ti|91911671, respectively. In both cases, the results were confirmed by PCR using different pairs of primers. The DNA is characterized as +/+, +/- and -/- by the presence of the interrupted allele. (C) A northern blot of total brain RNA from newborn mice of the +/+, +/- and -/- genotypes. This blot was probed with a mouse EST clone that was 94% identical over 284 bases to a region corresponding to the 3'-end of the human testis-specific RFX4 transcript H10145. The only visible transcript was of 4 kb (RFX4_v3); this was decreased in expression in the +/- sample, and undetectable in the -/- sample. Longer exposure of the blot did not reveal the presence of any truncated mRNA species in the +/- and -/- lanes. The same blot was hybridized to an actin cDNA (lower panel), and demonstrates roughly equivalent loading of the three RNA samples. (D) The hybridization of the same probe to adult mouse tissues, revealing an 4 kb transcript in brain (RFX4_v3), a 3.7 kb transcript in testis and a still smaller transcript in liver. (E) The pattern of developmental expression of the 4 kb transcript, which was undetectable in whole embryos at E7.5, highly expressed in whole embryos at E9.5 and 10.5, and less well expressed at E13.5 and 14.5. The brain, liver and testis lanes from D are juxtaposed in E to illustrate the difference in size between the brain (RFX4_v3), liver and testis transcripts, and the size identity of the adult brain transcript and the embryonic transcript. Also shown is the expression of a control mRNA for cyclophillin (Cyclo.).