A strategy to study tyrosinase transgenes in mouse melanocytes.
BACKGROUND
A number of transgenic mice carrying different deletions in the Locus Control Region (LCR) of the mouse tyrosinase (Tyr) gene have been developed and analysed in our laboratory. We require melanocytes from these mice, to further study, at the cellular level, the effect of these deletions on the expression of the Tyr transgene, without potential interference with or from the endogenous Tyr alleles. It has been previously reported that it is possible to obtain and immortalize melanocyte cell cultures from postnatal mouse skin.


RESULTS
Here, we describe the efforts towards obtaining melanocyte cultures from our Tyr transgenic mice. We have bred our Tyr transgenic mice into Tyr c-32DSD mutant background, lacking the endogenous Tyr locus. In these conditions, we failed to obtain immortalized melanocytes. We decided to include the inactivation of the Ink4a-Arf locus to promote melanocyte immortalisation. For this purpose, we report the segregation of the Ink4a-Arf null allele from the brown (Tyrp1b) mutation in mice. Finally, we found that Ink4a-Arf +/- and Ink4a-Arf -/- melanocytes had undistinguishable tyrosine hydroxylase activities, although the latter showed reduced cellular pigmentation content.


CONCLUSIONS
The simultaneous presence of precise genomic deletions that include the tyrosinase locus, such as the Tyr c-32DSD allele, the Tyr transgene itself and the inactivated Ink4a-Arf locus in Tyrp1B genetic background appear as the crucial combination to perform forthcoming experiments. We cannot exclude that Ink4a-Arf mutations could affect the melanin biosynthetic pathway. Therefore, subsequent experiments with melanocytes will have to be performed in a normalized genetic background regarding the Ink4a-Arf locus.
Lavado
Alfonso
A
Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, C/ Darwin, 3 28049 Madrid, Spain. Alfonso.Lavado@STJUDE.ORG
Matheu
Ander
A
Serrano
Manuel
M
Montoliu
Lluís
L
D000483:Alleles; D000818:Animals; D002460:Cell Line; D019941:Cyclin-Dependent Kinase Inhibitor p16; D008544:Melanocytes / Q000166:cytology* / Q000201:enzymology*; D008722:Methods; D051379:Mice; D008822:Mice, Transgenic; D014442:Monophenol Monooxygenase / Q000235:genetics*; D009154:Mutation; D019076:Transgenes; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics
C417743:Cdkn2a protein, mouse; D019941:Cyclin-Dependent Kinase Inhibitor p16; D025803:Tumor Suppressor Protein p14ARF; D014442:Monophenol Monooxygenase