Analysis of

sph^Dem/sph^Dem genomic DNA. (A) Top line, schematic of intron 10 and exon 11 of theSpna1 gene. Vertical lines denote exon/intron boundaries. Second line, normal sequence at the intron 10/exon 11 boundary; intron sequence is underlined. The IAP element present in thesph^Dem allele is shown on the third line, flanked by its LTR sequences (black boxes). The 6 bases preceding the IAP element on the third line represent exon 11 sequence duplicated by insertion of the element. Double hatch (//) marks denote sequence not represented in the figure. Filled and open triangles show the location of forward and reverse PCR primers, respectively. Sequence of PCR primers 67, 35, and 69r is found in the “Materials and methods” section; primer 69 is the complement of primer 69r. (B) Long PCR of genomic DNA from +/+ (lanes 2, 3), sph^Dem/+ (lanes 4, 5), and sph^Dem/sph^Dem(lanes 6, 7) mice with primers 67 and 35. M, marker lane, sizes of markers in kb are indicated on left. Top arrow on right identifies the 5.75-kb band resulting from amplification of the insert-containingsph^Dem allele of Spna1. Bottom arrow on right identifies the 0.35-kb band resulting from amplification of the normal allele of Spna1. (C) Genomic PCR of tail DNA from 2 different +/+ and sph^Dem/+ mice. Lane M is marker; sizes in kb are shown on left. Lanes ‘35’: PCR products from a reaction containing primers 67 and35 that will amplify the wild type allele of Spna1. Lanes ‘69r’: PCR products from a reaction containing primers (67 and 69r) designed to identify the IAP insertion in the sph^Dem allele. Genotype of mice is noted above each bracketed pair of reactions. (D) Thesph^Dem IAP is in the T subclass. Sequences of LTR elements derived from LS-type (‘L’, bottom line)24and T-type (‘T’, top line)25,26 IAP elements compared to that of the IAP element inserted in intron 10 of thesph^Dem allele of Spna1 (‘D’, middle line). The regions of sequence shown are used to classify IAP elements.24 Shaded bases represent sequence identity; nonshaded bases represent sequence divergence.