Antisense Treatment: Two
commercially available
Tbx2oligonucleotides
were used to block the sense
Tbx2(obtained from
Oligos Etc.). One antisense is complentary to the portion of
mRNA coding for the N-Terminus of the TBX2 protein called N-
Terminus. The other antisense is complentary to the portion
of mRNA coding for the Box (DNA binding domain) region of
the TBX2 protein, called Box antisense. The three
experimental culture conditions were as follows: 1)
N-Terminus antisense oligonucleotide; 2) Box antisense
oligonucleotide; 3) N-Terminus antisense oligonucleotide and
Box antisense oligonucleotide combined. The oligonucleotides
were introduced as a 200nM concentration in 0.66% Lipofectin
in OptiMEM (Life Technologies) as previously described
(Oligos Etc. protocol). The incubation in antisense occurred
at 37oC for four hours. The control condition contained 200
nM of sense Tbx2in
0.66% Lipofectin in OptiMEM.
RT-PCR:The
RNA was extracted from the explants by homogenizing the
cultured explants in phenolguanidium and precipitated as
previously described (Cebra- Thomas protocol). Reverse
Transcriptase was added to the extracted RNA to form cDNA.
PCR experiments were run for 35 cycles at an annealing
temperature of 65oC in the Minicycler apparatus using the
synthesized cDNA in the presence of buffer,
deoxyribonucleotide phosphates (dNTPs), and T. aquaticus
polymerase in a final volume of 50ml.
Three cDNAs were run in each of three different primers as
follows (cDNA/Primer):
|