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 immortalise 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 immortalised 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. Conclusion 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.


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 immortalised 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 (Tyrp1 b ) 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.

Conclusion:
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 Tyrp1 B 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.

Background
Eukaryotic genes are organised on chromosomes in units known as expression domains, that are believed to include all regulatory elements required for correct gene expression [1]. We use the mouse tyrosinase locus (Tyr) as an experimental model to study mammalian expression domains [2,3]. The mouse Tyr gene is located in chromo-some 7 [4], encodes the rate-limiting enzyme in melanin biosynthesis and is tightly regulated during development, being exclusively expressed in neural crest-derived melanocytes and optic cup-derived retinal pigment epithelium (RPE) cells [5,6].
Classically, the approach used to functionally identify regulatory elements has been testing a series of DNA constructs containing different amounts of regulatory sequences in transgenic animals. Minigene Tyr constructs were able to rescue the albino phenotype of recipient animals, but displayed variability in pigmentation [7,8]. In contrast, the generation of transgenic mice with a 250 kb yeast artificial chromosome (YAC) covering the entire mouse Tyr locus completely rescued the albino phenotype, resulting in mice that were indistinguishable from agouti wild-type pigmented mice [9,10]. These results pointed to the existence of important regulatory elements, absent in previous standard constructs, such as the locus control region (LCR), identified 15 kb upstream of the mouse Tyr promoter [11,12]. The LCR is necessary to establish the proper expression pattern of the mouse tyrosinase gene. The absence of the LCR resulted in weaker pigmentation, variegated expression in the melanocytes and RPE cells and delayed retinal pigmentation in transgenic mice [13]. Moreover, two binding boxes for nuclear factors within the LCR core, known as boxes A and B, were identified by in vitro analysis [14] and, recently, have been incorporated into a new boundary activity associated within the LCR region [15]. We have generated transgenic mice with new YAC Tyr transgenes carrying a range of specific mutations within the LCR region [10].
To address a more detailed study, both at the functional and structural level (using biochemical and cellular approaches), of the role of LCR-variants in these different transgenes, a number of problems had to be solved, including: (1), the dispersed nature of melanocytes which prevented us from direct analysis of relevant tissues, such as skin, where many other unrelated cell types are found; (2), the tyrosinase albino allele (Tyr c ), present in all recipient mouse strains used for the generation of transgenic mice, carrying a reported point-mutation within the coding region that results in a non-functional protein, without the transcriptional status of the locus being affected [16][17][18], and, [3], although it has been demonstrated that it is possible to obtain mouse melanocyte immortal cell lines from postnatal skins [19][20][21][22], often it becomes difficult to overcome the senescence period that all primary cell cultures undergo.
In this study we describe our efforts and the strategy to obtain melanocyte cell cultures from YAC Tyr transgenic mice in a genetic background lacking the endogenous mouse tyrosinase gene, and the effect of the inactivation of the Ink4a-Arf locus [23] on proliferation, senescence and tyrosinase activity of established melanocyte cell lines.

Results and discussion
Transfer of YAC Tyr transgenes from albino outbred NMRI mice (Tyr c / Tyr c ) to a Tyr c-32DSD / Tyr c-32DSD background Previous Tyr transgenic mice have been generated in albino outbred NMRI mice [9,10,12]. All observed pigmentation is due to the expression of the Tyr transgene but the presence of mutant albino Tyr protein and mRNA from the host Tyr c allele [16][17][18] could interfere in subsequent cellular, biochemical and genomic analyses. The albino 32DSD mutant mouse (Tyr c-32DSD ) carries a deletion of the entire Tyr locus, encompassing ~200 kb of mouse chromosome 7 [24]. Therefore, a breeding program was established between Tyr transgenic and albino 32DSD mice in order to obtain Tyr transgenes in animals lacking the endogenous Tyr alleles. Genotype analysis of the resulting mice was carried out by Southern blot. An EcoR I restriction fragment length polymorphism (RFLP) in exon 2 detected by the RFLP probe allows the identification of the transgenic Tyr allele (17 kb), derived from the mouse C3H strain, and the endogenous counterpart albino Tyr c NMRI allele (12 kb) (9) (Fig. 1).

Melanocyte primary cultures of YAC Tyr/∅ ; Tyr c-32DSD / Tyr c-32DSD
To gain further insight on a series of YAC Tyr transgenic mice carrying a range of deletions around the LCR [10,12], we decided to prepare cell lines that could be representative of these animals. Chromatin analyses cannot be done in tissue samples obtained directly from transgenic animals, due to the low number of cells expressing the Tyr gene (RPE cells and melanocytes) and the complexity of the tissues or organs containing these cells (eye and skin, respectively). In addition, the presence of the mutated, but transcriptionally active [16][17][18], albino Tyr locus in all transgenic mice generated to date could interfere with the interpretation and the acquisition of experimental data. To avoid this problem we mobilised the YAC Tyr transgenes to a genetic background lacking the endogenous mouse gene, as shown in Fig. 1, and then we tried to establish melanocyte cultures from these mice.
Mouse melanocyte immortal cell lines can be derived from postnatal skins [19][20][21][22]. Mouse crosses were established with parental lines to obtain pigmented pups with the desired genotype (YAC Tyr / ∅ ; Tyr c-32DSD / Tyr c-32DSD ). Genotype analysis was not necessary to distinguish between transgenic and non transgenic pups, because melanin, clearly visible in the eye and in the skin at this stage, could only derive from the YAC Tyr transgene expression. Melanocyte primary cultures from dorsal skin of heterozygous YAC Tyr transgenes in homozygous Tyr c-32DSD background were prepared as described [19]. Individual melanocytes and small melanocytes colonies appeared at culture day 10. The number of cells increased slowly until day 25-35, when melanocytes showed signs of senescence. Most cells died by day 85-90 and no immortal cell lines could be established ( Fig. 2A-C). Similar results were obtained from all YAC Tyr derivative transgenes bred to homozygous Tyr c-32DSD mice.
Mouse melanocyte primary cultures and their corresponding immortalised cell lines have been established from a number of mutant mice [25][26][27][28][29], although this type of cell lines can be sometimes difficult to achieve, as it may be inferred from these listed publications, in which reported cell lines have been generated by the same laboratory. A number of parameters can influence the success in obtaining immortalised melanocytes from mice. First, skins from mouse pups are used as starting material, carrying bacteria and other microorganisms that can contaminate the cultures. Second, and most important, melanocytes, as any somatic cell line in culture, undergo a senescence step previous to their immortalisation. Due to the low number or surviving cells after this senescence step, cells need to be cultured continuously during a minimum of 3-6 months to obtain an immortal cell line [19,20]. In most of the cultures we did not observe melanocytes after the senescence step ( Fig. 2A-C). These results were obtained with all different primary cultures, regardless of their genotype, indicating a problem at the immortalisation step.

Segregation of the Ink4a-Arf locus from the Tyrp1 b locus
It has been reported that melanocyte immortal cell lines (i.e. melan-a and melan-c) lack the p16 protein, most likely due to the lost of the Ink4a-Arf locus during the culture process [30]. Melanocytes from Ink4a-Arf (-/-) null mice proliferate exponentially without showing any signs of senescence, thus it has been proposed that the generation of melanocyte immortal cell lines in an Ink4a-Arf null background would be much easier [30]. Comparable results had been obtained before with fibroblast cultures from Ink4a-Arf homozygous mutant mice [31]. The absence of p16 leads to the inhibition in the inactivation of CDK4 and CDK6. These kinases inactivate the retinoblastoma pathway, promoting the proliferation of the cells [32,33]. Therefore, we decided to mobilise the Ink4a-Arf null allele into our YAC Tyr transgenic mice.
First, we had to remove the brown mutation co-segregating with the Ink4a-Arf null allele. The murine brown locus corresponds to the gene encoding the Tyrosinase-related protein 1 (Tyrp1), an enzyme which is also implicated in the melanin biosynthetic pathway [34]. It was originally noted that the coat colour of Ink4a-Arf null mice was paler than their wild-type littermates [23]. Indeed, biochemical evidence was presented that was highly suggestive of defective Tyrp1 activity in Ink4a-Arf null melanocytes [35]. Homozygous Tyrp1 b mutant mice display less tyrosinase activity than wild type, because mutated forms of the Tyrp1 protein affect tyrosinase processing [36]. Notably, the Tyrp1 locus, is close to the Ink4a-Arf locus, in mouse chromosome 4, at a distance of 8.7 Mb (mouse ENSMBL, build 32). Most inbred laboratory strains with defective Tyrp1 activity carry the recessive brown allele, Tyrp1 b that contains a single amino acid change at a critical residue in the Tyrp1 protein [37]. To test directly whether the Ink4a-Arf null allele was linked to the Tyrp1 b allele, two single nucleotide polymorphisms (SNPs) that characterize the Tyrp1 b allele and that can be diagnosed by subsequent restriction enzyme digestion [37] were used. In particular, a Tyrp1 b -linked SNP at exon 4 eliminates a Taq I restriction site and, similarly, another Tyrp1 blinked SNP at exon 5 eliminates an Hga I restriction site. Using these two markers, we directly proved that the original Ink4a-Arf null mice were indeed homozygous for the Tyrp1 b allele, thus explaining their paler coat colour (Fig.  3). The Tyrp1 b recessive allele linked to the mutated Ink4a-Arf allele was probably derived from the ES cell line originally used for targeting the Ink4a-Arf locus, namely WW6 ES cells, which had a complex genetic background [38].
The absence of at least one Ink4a-Arf allele overcomes the senescence of primary melanocytes in culture Ink4a/Arf-null strain in a pure C57BL/6J genetic background and unlinked from the mutant Tyrp1 b allele were obtained to avoid the confounding presence of the Tyrp1 b allele. Ink4a-Arf +/mice were backcrossed seven times with wild-type C57BL/6J mice, eventually yielding Ink4a-Arf +/-; Tyrp1 B / Tyrp1 b mice. These mice were intercrossed to produce a number of Ink4a-Arf -/mice that were in their majority Ink4a-Arf -/-; Tyrp1 b / Tyrp1 b and, accordingly, had a brown coat. Exceptionally, one mouse (from a total population of 27 Ink4a-Arf -/mice) was identified as an Ink4a-Arf -/but had a black coat. This mouse turned out to represent a recombinant with an Ink4a-Arf -/-; Tyrp1 B / Tyrp1 b genotype. From this animal, and after the appropriate crosses, a strain of mice in C57BL/6J background that were Ink4a-Arf -/-; Tyrp1 B / Tyrp1 B was obtained (Fig.  3), and used for subsequent experiments.

Melanocyte primary cultures from Tyrp1 B / Tyrp1 B Ink4a-Arf mutant mice
To evaluate the effect of the inactivation of the Ink4a-Arf locus in Tyrp1 B / Tyrp1 B genetic background on the immortalisation of mouse melanocytes, melanocyte primary cultures from dorsal skin of Ink4a-Arf +/+ , Ink4a-Arf +/-, Ink4a-Arf -/pups in a Tyrp1 B background were prepared (Fig. 2D-L), using the same described procedures [19]. Individual melanocytes and colonies appeared at day 10. The number of cells increased slowly in Ink4a-Arf +/+ (Fig.  4A) and, around day 35, melanocytes increased their size and lost their bipolar shape. Few or none Ink4a-Arf +/+ melanocytes survived the senescence step, and most of these melanocytes died by day 85-90 ( Fig. 2D-F). In contrast, Ink4a-Arf +/and Ink4a-Arf -/kept their bipolar shape and small size, with a limited number of cells showing any sign of senescence ( Fig. 2G-L). Proliferation of Ink4a-Arf -/melanocytes was higher than in Ink4a-Arf +/melanocytes, and both were much higher than in Ink4a-ARF +/+ melanocytes (Fig. 4A). By day 45 of culture, Ink4a-Arf -/melanocytes entered the exponential phase of growth. In contrast, Ink4a-Arf +/melanocytes did not show evidences of exponential growth until day 65 (Fig. 4A). This delay could be explained by LOH (loss of heterozygosity), spontaneously occurring in Ink4a-Arf +/melanocytes and affecting the remaining wild-type allele of the Ink4a-Arf locus, a common event that is known to take place both in vitro [39] and in vivo [40]. Similar results have been reported [30] using independent mouse colonies.

Tyrosinase activity and melanin content in the melanocyte cultures from Ink4a-Arf mutant mice
A valid approach to analyse the role of the different regulatory regions of the mouse Tyr gene in the transcription of the locus and, eventually, in the amount of mature protein being made, is the measurement of the enzymatic activity of the derived Tyr protein. We measured the levels of Tyr enzymatic activity and melanin content using reported procedures [41] in melanocyte cultures, to study Generation of an inbred Ink4a/Arf -/-; Tyrp1 B / Tyrp1 B mouse strain . Bars represent the mean value (+/-SD) from three replicates. Tyrosinase activity and melanin content were measured in melanocyte primary cultures at passage 8 (~day 70 of culture). Statistically significant differences (t-Student test) are indicated as follows: * p < 0,1; ** p < 0,01; *** p < 0,001. the influence of the presence or absence of the p16 protein in the expression of the Tyr gene. Tyrosine hydroxylase activity values from Ink4a-Arf -/and Ink4a-Arf +/cell extracts were undistinguishable and significantly lower to values obtained in melan-a cells (Fig. 4B). However, the quantity of melanin in the proliferative Ink4a-Arf -/melanocytes cell cultures was significantly lower than in Ink4a-Arf +/or melan-a cells (Fig. 4C).
Differences in melanin content could be explained by different individual cell culture response to TPA or/and CT that are present in cell culture medium. However, the observed differences in cellular pigmentation were maintained after removing CT from cell culture medium. In addition, TPA is always required as an additive to maintain cellular proliferation. Increasing the amount of TPA does not result in a parallel increase in pigmentation, opposite to what is observed with CT.
Differences in melanin content could also be explained by the control of the retinoblastoma (RB) protein by p16, and the observation that RB protein interacts with the transcription factor microphthalmia (Mift) [42], that controls the expression of the Tyr gene. Differences between Ink4a-Arf mutant cells and melan-a could be due to the presence of a number of additional alterations in this latter immortal cell line, such as the loss of expression of p16Ink4a [30]. The recent generation of mice with increased gene dosage of Ink4a-Arf will be instrumental to further investigate the influence of this locus on Tyr activity [43].

Conclusion
With all these results we can conclude that the simultaneous presence of: [1] at least one mutant allele of the Ink4a-Arf locus; [2], the Tyr c-32DSD mutant albino allele in homozygosis and; [3], the presence of the relevant Tyr transgene in heterozygosis, are required for the establishment and the study of immortal mouse melanocyte cultures from transgenic mice carrying Tyr constructs. Finally, we cannot exclude that Ink4a-Arf mutations could affect the melanin biosynthetic pathway. Therefore, experiments with mouse melanocytes will have to be performed in a normalized genetic background regarding the Ink4a-Arf locus.

Southern blot analysis
The discrimination of the endogenous tyrosinase gene from the YAC-tyrosinase transgenes was performed as previously described, using the RFLP probe, containing exon 2 of the mouse tyrosinase gene [9,12]. To obtain an endogenous internal control, membranes were co-hybridised with a single-copy mouse gene, the p19 Arf E1 probe, a EcoR I DNA fragment, 230 bp in length, containing exon 1 of the p19 Arf gene (pRSp19arfE1 plasmid, generous gift from M. Malumbres). In brief, genomic DNA was isolated from mice tail tips and prepared for southern blot as described [12]. 15-20 µg of genomic DNA were digested with EcoR I (Roche, Basel, Switzerland), fractionated by horizontal gel electrophoresis in 0,8% agarose and transferred to a Hybond-N nylon membrane (Amersham, Buckinghamshire, UK) by capillary blotting. RFLP and p19 Arf E1 DNA probes were labelled with [α 32 P] dCTP using the High Prime labelling kit (Roche). Membranes were hybridised in Southern hybridisation solution (0.25 M Na 2 HPO 4 pH = 7.2, 7% SDS, 1% BSA) overnight at 65°C, washed at 65°C in 20 mM Na 2 HPO 4 pH = 7.2, 1% SDS, 1 mM EDTA pH = 8 and resulting blots exposed for 1-3 days and scanned with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA). Quantification of the hybridisation signals was performed using the ImageQuant v1.2 software (Molecular Dynamics).

Melanocyte cultures
Melanocyte cultures were prepared, essentially, as previously described [19]. Briefly, dorsal skin biopsies were obtained from pups of all investigated genotypes between +19.5 and +22.5 d.p.c. stages (postnatal P2-P3). Dorsal skin was split in 5 µg/ml trypsin (Sigma, St. Louis, MO, USA) in PBS and the epidermal layer then minced with a pair of surgical blades in 250 µg/ml trypsin and 200 µg/ ml EDTA in PBS. Cells were cultured on a feeder layer of mitomycin-treated murine XB2 keratinocytes [44]. The cells were grown in RPMI-1640 medium containing 2 mM glutamine, 10% fetal calf serum, 100000 U/l penicillin, 100 mg/l streptomycin sulphate (all from Invitrogen, Carlsbad, CA, USA), 200 nM tetradecanoyl phorbol acetate (TPA) (Sigma) and 200 pM cholera toxin (CT) (Sigma), at 37°C, 95% humidity and 10% CO 2 pressure. Explant cultures from different donor mice were kept separate. Passages were made when cultures became subconfluent, and melanocytes were counted at each passage. Feeder cells were added when necessary. Control melan-a and melan-c cells, derived from inbred C57BL/6J and outbred albino LAC-MF1 mice, respectively, were cultured as previously described [19,20].

Quantification of melanin and tyrosinase enzymatic activities
Melanin contents in whole cell extracts were measured by spectrophotometer essentially as described [45]. In brief, 6 × 10 6 cells from ~day 70 of culture were collected and homogenised in 300 µl of PBS, 100 µl of homogenate incubated for 14-16 hours at room temperature with 900 µl of 2 M NaOH, 20% DMSO and absorbance measured at 470 nm.