LÍNEA POMASQUI – CRISTIANÍA 138 kV (CASO 3)

All oligonucleotides used were synthesised by Genosys Biotecnnologies Ltd. (Cambridge), with a phosphorothioate backbone to aid stability in-vitro.

2.4.1

C-myc antisense and ‘4G ’ quartet control sequences

The c-myc antisense sequence was a 15-mer ODN complementary to the translation- initiation codon of exon 2 o f the c-myc gene. The ‘4 0 ’ quartet control was also a 15- mer ODN. This was not complementary to the c-myc gene sequence but contained 4 guano sine residues which are known to produce non-specific effects on cell growth in some experiments. This was therefore the most important control sequence to use. Neither sense nor nonsense ODNs had previously been shown to effect the growth of the A375m cell line in-vitro (Jagdeep Ghana thesis 1998) and were therefore not used in these experiments. The sequences of the c-myc antisense and the ‘4 0 ’ control are shown in Table 2.1.

Phosphorothioate Oligonucleotide Sequence

C-myc antisense 5 ’ - AACGTTG AGGGGC AT-3 ’ ‘4G ’ Quartet Control 5 ’ - AAGC ATACGGGGTGT-3 ’

Table 2.1 C-myc antisense and ‘4G’ quartet ODN control sequences

2.4.2

Preparation and storage of oligonucleotides

Oligonucleotides were purified by high-pressure liquid chromatography (Genosys Biotechnologies Ltd). The products were supplied lyophilised and were dissolved at a concentration of 0.5nm/pl in sterile phosphate buffered saline containing 20mM/l

of HEPES buffer. Prepared aliquots containing 20pl of dissolved oligonucleotide were stored at -20^C in eppendorf tubes until required.

2.5

Targeting the c-myc gene using a ribozyme

2.5.1

Target and structure of the c-myc ribozyme

A ribozyme was designed according to standard principles to target the same translation-initiation codon of exon 2 of the c-myc mRNA which is targeted by the 15-mer c-myc ODN (base pairs 296-311). The efficacy of this c-myc antisense ODN has been demonstrated in melanoma by Jagdeep Ghana (MD thesis 1998) and by Citro and Leonetti both in-vitro and in-vivo (Leonetti et al. 1996, Citro et al. 1998).

A -GUU- triplet is present at the 3 ’ end o f the translation-initiation codon o f exon 2 (base pairs 303-305) of the c-myc mRNA. This triplet sequence is known to be a cleavage site for ribozymes and the structure o f a hammerhead ribozyme was therefore designed around this site (figure 6.2). The standard -AAAG- and - CUGAUGA- sequences, along with a sequence high in guanine and cytosine residues, were selected to construct stem II of the ribozyme. A sequence of 5’- GAAGCU-3’ was complementary to the base sequence on the 3 ’ side o f the -GUU- triplet and formed stem I. A 5’-GUUGAGG-3’ sequence was complementary to the base sequence on the 5’ side of the -GUU- triplet and formed stem II.

The plasmid pREV, previously constructed by Spencer Collis at the Paterson Institute o f Cancer Research in Manchester, was selected as a vector to express the ribozyme targeting c-myc. The structure o f this plasmid is shown in figure 6.3. The plasmid contains Green Flourescent Protein (GFP) as a green fluorescent marker whose expression is promoted by a CMV I.E. promoter. It also has a SV40 polyadenylation site at the 3’ end to stabilize the RNA transcript in mammalian cells. A ribozyme-cloning site is present, with a CMV promoter and SV40 polyadenylation site at the 3 ’ end, to promote ribozyme expression. The plasmid also contains genes encoding ampicillin and neomycin resistance whose expression is promoted by SV40 promoters.

2.5.2

Production of the ribozyme-encoding plasmid

targeting the c-myc oncogene

2.5.2.1

Annealing of oligonucelotides

The first stage in the production of the ribozyme was annealing o f the two oligonucleotides MycRzA, encoding the ribozyme, and a complementary oligonucleotide MycRzB. Both oligonucleotides were 41 bases in length and their sequences are represented in figure 6.4. Annealing of the oligonucleotides occurred with a 4-base overhang at each end such that a double-stranded base sequence resulted with two single-stranded ends (see figure 6.4). The single-stranded terminae were complementary to ih^Xba-l and Mlu-1 cloning sites in the vector.

The two ODN’s were mixed together at a ratio o f 1:1 at two different concentrations (lOOpmol and 250pmol) in lOpl of distilled water. The mixture was heated at 55°C for 5 minutes and then cooled to allow annealing to occur, and then left overnight.

2.S.2.2

Ligation

Before the annealed nucleotide sequences could be ligated into the pREV plasmid vector, the pREV circular plasmid DNA was digested to yield a linear, double­ stranded DNA sequence to which the annealed nucleotides could be ligated and thus reform the circular loop. After digesting the plasmid to form the linear sequence, each end of the plasmid DNA was digested to form ‘sticky’ ends to which the annealed nucleotides would bind.

Linearisation of the pREV plasmid

The pREV plasmid has àXba-1 cleavage site which is the ribozyme cloning site. To cleave the circular plasmid DNA, 10 units of Xba-J enzyme (MBI Fermentas) was therefore added to 4ug o f pREV DNA (in 5pi of distilled water), lul of buffer Y

(MBI Fermentas) and 3ul o f distilled water and the solution incubated at 37°C for 2 hours. This yielded a linear plasmid DNA sequence with 2 Xba-1 terminae.

The mixture then underwent a clean-up stage:

1. 100pi of PN buffer (QlAgen) was added to the DNA digest to allow binding to the resin in the QlAquick centrifuge columns (QlAgen) and the mixture added to the QlAquick columns in centrifuge tubes.

2. The tubes were centrifuged at 6500rpm for 1 minute to allow filtration. The plasmid fragments remained in the resin.

3. 750pl o f buffer PE (QlAgen) was added and the tubes spun again at 13000rpm for 1 minute to wash the final salts off the DNA fragments. A second spin at 13000rpm for 1 minute was used to wash off the alcohol.

4. 100pi o f elution buffer (QlAgen) was then added to the column for 1 minute and the column centrifuged at 13000rpm for a further minute to remove the plasmid DNA fragments from the resin.

5. One end of the linearised DNA sequence was then digested by the Mlu-1 enzyme. 2pi (20 units) of Mlu-1 enzyme solution (Fermentas) was added to 5pg of linearised pREV DNA (in 5pi o f distilled water), with 2pi of buffer R (Fermentas) and 11 pi of distilled water. This yielded linearised pREV DNA with a. Xba-1 binding site at one end and a Mlu-1 binding site at the other. These were complementary with the Xba-1 and Mlu-1 binding sites which were present at either end of the annealed nucleotide sequences.

6. A second clean-up stage was then followed for the Mlu-1 digested plasmid DNA using the same method as described above.

7. A lOpl portion of the elute was then run on an electrophoresis gel to establish the concentration o f plasmid DNA in the solution. The elute contained linearised plasmid DNA at a concentration of 50ng/pl. The size of the linearised DNA fragment was 7.8kb.

Ligation reaction

Addition of the annealed nucleotide sequence to the linearised plasmid sequence, with the Xba-1 and Mlu-1 ends, resulted in ligation of the nucleotide sequence into the pREV plasmid and the reformation of the circular plasmid DNA, incorporating

the ribozyme-encoding nucleotide sequence. The digested plasmid was then mixed with the annealed ODN’s in the presence of ligase enzyme and ligase buffer to allow ligation to occur. A range of concentrations of both DNA reagents was used to ensure at least one of the ligation reactions was efficient. Either 200ng of linearised pREV plasmid in 4pi o f distilled water or 350 ng o f linearised pREV plasmid in 7pi of distilled water was mixed with the annealed ODNs. 2pi of ligase buffer, Ipl of ligase enzyme and 3 pi of distilled water were used in the first case to make a total of lOpl, however no water was added for the second, higher concentration. The two concentrations of ODNs used were either 0.1 nM of each ODN or 0.25nM. There were therefore 4 different mixtures resulting. The solutions were incubated overnight at 22®C to allow ligation to occur.

In addition, 4 control reactions, containing the single-chain ODN c-mycRzA instead o f the annealed ODN’s, were set up paralleling each of the 4 combinations as described above. These were used to assess which reaction was the most efficient - ie to assess which reagent concentration combination produced the most efficient ligase enzyme effect. After bacterial transformation, plating and incubation, the concentration combination that produced the best ratio of colonies on the positive plate in comparison to the negative one, was the combination at which the enzyme had worked best. Isolation and PCR screening of bacterial colonies from this positive plate was therefore most likely to yield the highest chance o f obtaining successfully transformed clones.