Flanking genomic region of Tyr::Cre mice, rapid genotyping for homozygous mice Sophie Colombo, Vale ´ rie Petit   , Mayuko Kumasaka, Ve ´ ronique Delmas and Lionel Larue* Developmental Genetics of Melanocytes, UMR 146 CNRS, Institut Curie, Bat. 110, 91405, Orsay Cedex, France *Address correspondence to Lionel Larue, e-mail: lionel.larue@curie.fr   Present address: UMR 144 CNRS – Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 5, France. doi: 10.1111/j.1600-0749.2007.00387.x Dear Sir, Specific gene modifications in melanocytes can now be introduced with the Cre ⁄ LoxP system (Delmas et al., 2003). The Cre ⁄ LoxP site-specific recombination system of bacteriophage P1 is a powerful and accurate tool enabling conditional and tissue-specific manipulation of genes in mammalian cells both in culture and in mice (Gu et al., 1993; Nagy and Mar, 2001). We have previ- ously established a mouse line producing the Cre recombinase in the melanocyte lineage using the tyros- inase promoter (Tyr::CreA) from embryonic day 10.5 (Delmas et al., 2003). We occasionally detected Cre transgene activity in the brain and peripheral nerves in this mouse line. Generating specific null mutations in essential genes, including E-cadherin, b-catenin and Igf1r in melanocytes did not lead to lethality (unpub- lished results). Nevertheless, affecting the expression of other genes may produce a phenotype that is unrelated to coat color (unpublished). Discrimination of homozygous and hemizygous Tyr::Cre mice should increase the rate of mutant pro- duction and decrease the amount of time-consuming work involved. Whereas this distinction cannot be made rapidly by classical genetic techniques (despite our having shown that the Tyr::Cre transgene is located on the X chromosome), molecular genetic techniques can be useful. We initially estimated homozygosity and hemizygosity of the Tyr::Cre trans- gene by Southern blot analysis and by semi-quantita- tive PCR (Q-PCR). We found Southern blot analysis to be time consuming with modest accuracy and Q-PCR analysis to be expensive with poor accuracy. Deter- mining the flanking region of the transgene is the most efficient screening method, requiring the identifi- cation of the transgene location in the genome. The most efficient way to screen mice now is the method previously reported for Dct::LacZ mice (Takemoto et al., 2006). We improved mouse production efficiency by clo- ning the site of integration and generating oligonucleo- tides that can be used to identify homozygous (Tyr:: Cre ⁄ Tyr::Cre = X tg ⁄ X tg ), hemizygous (Tyr::Cre ⁄ X= X tg ⁄ X°) and wild type (X°⁄ X°) female mice. We diges- ted one flanking region of the Tyr::Cre transgene with Avr II (Figure 1) and carried out self ligation at a low DNA concentration (about 1 ng ⁄ ll). Use of this DNA concentration avoided concatemer production. We amplified the circularized DNA by PCR with LL1325 Figure 1. Principle of amplification and sequencing of the Tyr::Cre transgene insertion site. Top panel: schematic representation of the Tyr::Cre transgene in the Tyr::CreA mouse genome. Bottom panel: schematic representation of the self-ligated fragment after digestion with Avr II. The genomic DNA is represented by a double horizontal line and the tyrosinase (Tyr) enhancer, by a gray to white rectangle. The numbers in brackets correspond to the distances between the 5¢-end of the transgene and Avr II restriction sites. LL1326 and LL1325 primers were used to amplify and sequence the site of integration of the transgene. (LL1326: 5¢-CAG CAG ACA CCA AGG AAA CA-3¢) and (LL1325: 5¢-TGT TCC CTT TTG GTT TCA GG-3¢). The schemes are not shown to scale. ª 2007 The Authors, Journal Compilation ª 2007 Blackwell Munksgaard 305 Pigment Cell Res. 20; 305–306 LETTER TO THE EDITOR