In order to optimise the screening conditions, we reconstructed the experimental procedures using genes known to alter melanogenesis in melanocytes. The genes used
these genes as positive controls to test the system. First, Ras being an oncogene, we
were testing our system for its ability to detect genes involved in melanocyte
transformation. Second, using Agouti, we were testing our system both for its ability
to detect secreted molecules affecting pigmentation, and for molecules affecting the pigment quality and its production levels. Third, by using genes affecting melanocyte biology in very different ways, we were testing both the validity o f using
pigmentation as the discriminating marker and the versatility o f our system. In both cases, depigmentation of melanocytes was visible within ten days after infection. This was a very important observation because it gave us an indication o f the minimum amount of time we had to wait before we could start detecting pigmentation changes. Fortunately, ten days was about the time needed for selecting the successfully infected cells with Hygromicin. Moreover, once selection was over, cells were seeded at low density for colony formation, so that the visual screening for unpigmented colonies was beginning at least 24 days after infection o f the cells, giving enough time for the phenotype to develop.
Melan-a infected with pHMII-Agouti were also used to optimise the provirus
recovery. The MaRX system is characterised by the capacity o f recovering the cDNA transduced into the target cells through a specific CRE recombinase reaction. The efficiency o f the recovery depends on two main factors: the activity o f the CRE recombinase enzyme and the quality of the substrate, i.e. the target genomic DNA. When performing a screening, it is important to maximise the chances o f recovering potential positive genes and therefore we decided to optimise both the expression and
purification of CRE recombinase from bacterial cells, and the genomic DNA preparation. Expression o f the enzyme was greatly enhanced by changing the
conditions from 4h at 37°C to 16h at RT. Indeed, it is known that a lower expression temperature leads to slower protein synthesis and this in turn can help with the folding and solubility of recombinant proteins. We also optimised the sonication steps in order to maximise the recovery of the recombinant protein. Sonication is an efficient way to lyse cells in the non-denaturing conditions necessary for the recovery o f an active protein. On the other hand, extensive sonication can be disruptive to the protein and it is therefore important to find the right balance between efficient lysis of the cells and protein preservation. We found that for our purposes it was important to keep the cells cold at all times, and by sonicating the sample five times for 1 minute with two minutes rest, we obtained a high yield of active recombinase. This procedure proved successful, as shown assessing the activity o f the purified recombinant protein on a control vector.
The CRE recombinase purified as described above was used to recover the pro virus from Melan-a cells infected with pHMU-Agouti. In parallel, as a positive control, we performed the provirus recovery from fibroblasts infected with the same construct. It soon became obvious that the different origin of the genomic DNA was having a strong effect on the efficiency o f the reaction. Indeed, using the same conditions for genomic DNA extraction and subsequent CRE recombinase reaction, we could recover the pro virus from the control fibroblasts but were unable to do so from Melan-a. The main difference between the two cell lines was the presence of melanin
in the melanocyte sample. Indeed, even when melanocytes are depigmented because
of the overexpression o f particular genes like Agouti or Ras, there is always some
remaining melanin left in the melanosomes. This residual pigment, normally confined into specific organelles, upon lysis of the cells is released and can associate with nucleic acids and proteins. This “sticky” property o f melanin has been reported by different groups and has been shown to inhibit many enzymatic reactions like PCR
and reverse-transcription (Giambemardi et al, 1998; Eckhart et al, 2000). It was
therefore plausible to think that a similar inhibitory effect was the cause o f the poor efficiency of pro virus recovery observed for Melan-a samples. In order to remove the residual melanin, we optimised the extraction and designed a different method to isolate genomic DNA from melanocytes. First, cells were treated with 200pM PTU for one week before genomic extraction. PTU is a reversible inhibitor of Tyrosinase, the first enzyme involved in melanin synthesis, and is therefore able to reduce
pigment levels in melanocytes. This was followed by extraction o f the genomic DNA using the QIAGEN genomic DNA Kit, which includes a step to separate the cells nuclei firom the remaining organelles reducing the possibility o f melanin associating to the DNA. Finally, the genomic DNA was further purified with three
phenol/chloroform extractions. At the end o f the process, the DNA isolated visibly contained less pigment than the one obtained with previous methodologies. Indeed, when the DNA thus purified was treated with CRE recombinase, the efficiency of provirus recovery was greatly enhanced and similar to the one observed with control fibroblasts, validating the efficacy of this method.