Construction and characterization of a transgenic mouse expressing the <i>hMTH1</i> cDNA.

<p>(A) A BamH1-EcoRV fragment (509 bp) derived from pcDEBΔ <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000266#pgen.1000266-Kang1" target="_blank">[23]</a> encoding the <i>hMTH1</i> cDNA was subcloned into the gWIZ vector under the control of the CMV promoter. This vector transfected into wild-type MEFs expressed the hMTH1 protein (data not shown). The MscI-KpnI fragment (2481 bp) was used in the construction of the transgenic mouse. (B) Determination of transgene copy number. Genomic DNA from mouse tails was analysed by Southern blot analysis (left panel). In the right panel 2, 5, 10, 20, 40 copies of the MscI-KpnI fragment were analysed. Both blots were probed with the same probe. Copy number was determined by comparison. The arrow indicates the DNA from the mouse that was used as a founder for the colony. (C) Analysis by FISH of the number of hMTH1 integrations in hemizygous (<i>hMTH1-Tg^+/−</i>) and homozygous (<i>hMTH1-Tg^+/+</i>) strains. (D) Expression of <i>hMTH1</i> mRNA. RT-PCR was performed using total RNA from the indicated organs and human specific primers. RT-PCR for the <i>GADPH</i> gene is used as an internal control. <i>hMTH1</i> and <i>GAPDH</i> fragments are respectively 200 bp and 330 bp. (E) Western blot analysis of transgene expression. Total proteins (20–40 µg) from a range of tissues were separated by SDS polyacrylamide electrophoresis, blotted and probed with an antibody against hMTH1. β-tubulin was used as a loading control.