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One o f the key advantages of using the pHMII system is that, upon selection and expansion o f the positive clones, the cDNAs responsible for the observed phenotypes can be recovered for further characterisation.

To do so it is necessary to extract the cells’ genomic DNA, in which the cDNA of interest is integrated, and treat it with CRE recombinase. This causes the cDNA to be excised from the genome in the form of a plasmid that can in turn be transformed and expanded in bacteria.

Melan-a S Ï- ••> * / C iv C. OÏ>v. '■ k u ,‘ V ' ^ A ./ V '. P .V ' , o x>- VV . 0 ^ 0 Melan-a/Agouti c # ■ 9 K- ■ J ‘ \ i ' -A _'f i \ : 1' ' - ^ W o • 0 _{o ' ^} O f _ ' Ï ' ' ' 7 o j ' ; ' / - n r f ' ' "9 4" - Melan-a/Ras

Fig 3.7 Phenotypic changes induced by Agouti or Ras when overexpressed in

Unstained J»’ * ^ ^ . ' • A î ' - '>■''■■ pHMII pHMII-Agouti Crystal Violet Staining

m .%

Fig 3.8 1500 cells of Melan-a infected with vector only (pHMII) or vector expressing

the Agouti cDNA (pHMII-Agouti) were plated on a 10cm dish for 14 days. A) Unstained plates: only the pigmented colonies are visible

B) Same plates stained with Crystal Violet dye: both pigmented and unpigmented colonies are visible

The first thing we decided to optimise was the CRE recombinase expression and purification in bacteria. Our laboratory had access to the CRE cDNA cloned into the pET19 expression vector. The standard protocol in use included an expression step at 37°C for 4h. This was followed by purification through a metal-chelating resin, the recombinant protein being expressed with a His-Tag in the N-terminus. This method, although providing enough CRE activity for a few reactions, was limiting in the amount o f protein produced, indeed below the resolution limits of a standard Coomassie-Blue staining. By changing the expression conditions to RT for 16h and by optimising the sonication step on the bacteria, we were able to purify high amounts of protein (Fig 3.9). Moreover the enzymatic activity o f the protein was assessed using the control DNA provided by Novagen when purchasing their commercially

available CRE recombinase. The control DNA is a linearized X phage vector whose

core is made of a pET based sequence (with Ampicillin resistance gene) flanked by two LoxP CRE recombination sites.

When the DNA is treated with CRE recombinase, the recombination between the two LoxP sites causes the pET plasmid to be excised and circularised. If the DNA so treated is transformed into bacteria, the number o f Ampicillin resistant colonies observed will be proportional to the CRE reaction efficiency. In our test, using 50ng

of X vector DNA, we had 52 colonies in the negative control (X vector with no CRE),

207 colonies using Ipl of Novagen CRE and more then 2500 colonies using Ipl of CRE purified in our laboratory.

MW 1 2 3 4 5 6

His-tagged CRE

Fig 3.9 His tagged CRE recombinase was purified on metal-chelating resin. Six

SOOpl elutions were collected and added to SOOpl of 100% glycerol. lOpl of each elution were run on 12% polyacrylamide gel. The position of

recombinant CRE recombinase (~40Kda) is indicated. Elutions 2, 3, and 4 were stored at -80°C.

The reconstruction on the genomic DNA extracted from mammalian cells was carried out on genomic DNA from 5x10^ Melan-a cells infected with pHMII-Agouti. The DNA was purified using a standard protocol consisting o f lysing the cells in lysis buffer containing 1% SDS followed by phenol/chloroform extractions. The DNA thus prepared was treated with CRE recombinase and transformed into bacteria.

Unfortunately no colonies were observed suggesting that the CRE recombinase reaction was not successful for this sample. On the contrary a control reaction on genomic DNA extracted from fibroblasts, worked within the efficiency limits o f the technique (~1 bacteria colony per 5,000 mammalian cells). This was probably caused by the presence of melanin in the genomic DNA preparation from melanocytes. When cells are lysed, the residual amount of pigment present in the melanosomes is released and can unspecifically associate to nucleic acids and proteins. This is known to inhibit

enzymatic reactions such as PCR and reverse-transcription (Giambemardi et a/, 1998;

Eckhart et al, 2000). It is therefore plausible that melanin, even at the low

concentrations present in unpigmented looking melanocytes, will inhibit any enzyme that has to interact with DNA such as CRE recombinase.

To overcome this problem a new protocol for extracting genomic DNA from melanocytes was developed.

Cells were treated for a week prior to extraction with 200pM PTU (Phenylthiourea, an inhibitor of Tyrosinase) to further reduce their pigment content.

Moreover, the genomic extraction was carried out using QIAGEN genomic DNA columns following the manufacturer’s instructions. These include a step to isolate cell nuclei from cell debris, hence reducing the possibility o f melanin interacting with

DNA. Finally, genomic DNA was further purified by Phenol/Chloroform extractions and ethanol precipitation.

This protocol proved successful, and the CRE recombinase reaction efficiency on genomic DNA from melanocytes thus prepared was similar to the control one.