Generation of <i>Col2a1-Dlx5</i> transgenic mice.

<p>(A) Transgene design. Numbering of promoter and intron elements is with respect to the <i>Col2a1</i> transcription start site. The start codon of <i>Col2a1</i> in exon 1 has been mutated to prevent translation in this exon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008097#pone.0008097-Zhou1" target="_blank">[56]</a>. The asterisk indicates the <i>Flag</i> sequence 5′ to the murine <i>Dlx5</i> open reading frame, bpA, poly-adenylation sequence from the bovine <i>Growth Hormone</i> gene; SA, splice acceptor. Half arrows indicate the approximate location of primers for genotyping: Col2a1 forward (Cf) plus Dlx5 reverse (Dr), and for RT-PCR: Flag forward (Ff) plus Dr. (B) Transgene copy number. Semi-quantitative PCR was used to compare the approximate transgene copy number in transgenic founder mice: t2/+ and t3/+ represent dead hemizygous founders and t19/+ is a hemizygote neonate from the stable <i>Col2a1-Dlx5^t19/+</i> line. Control lanes from the left are: wild type DNA (0), wild type DNA mixed with <i>p3000i3020Col2a1-Dlx5</i> at 1 copy per genome equivalent (1) or 10 copies per genome equivalent (10). Amplification of a genomic fragment of the single copy gene <i>Ihh</i> was used to judge relative amplification of the transgene. (C) Specific amplification of expressed <i>Flag-Dlx5</i> sequence from transgenic embryos in a reverse transcriptase-dependent manner. RT-PCR analysis of five F4 generation embryonic day (E) 17.5 embryos from a <i>wt</i> x <i>Col2a1-Dlx5^t19/+</i> mating demonstrates stable heritable expression of the <i>Col2a1-Dlx5</i> transcription unit. The first lane in the bottom panel (+) shows a positive PCR control for the minus RT experiment. Non-adjacent lanes from the same gel have been spliced together to generate the figure. Lane numbers refer to individual embryos.