Transient transfection of
the TPO gene and TPO cDNA constructs into the Hep3B cell line. (A) Schematic representation of the human TPO gene
(GenBank accession no. D32046) and TPO cDNA minigenes cloned in
pcDNA 3 vector. E (white boxes), exons; I (lines), introns; black
box, the 116 nt alternative intron. Sizes of exons and introns are
indicated. KpnI and NotI are the
cloning sites. ATG and TAA, start and stop codon, respectively.
(B) Analysis of pre-mRNA splicing of human constructs
in Hep3B cells. The upper band corresponds to the full-length TPO
cDNA (1109 bp) and the lower band to the TPO-3 cDNA lacking the116
nt sequence (993 bp). Amplicons were separated on 0.8% (w/v)
agarose gel. Endogenous TPO poly(A), RT–PCR of RNA derived
from non-transfected Hep3B cells retrotranscribed with poly(dT)
primer. TPO gene and TPO cDNA, RT–PCR of RNA from transfected
Hep3B cells retrotranscribed with pcDNA reverse primer, specific
for TPO constructs. Endogenous TPO pcDNA rev, RT–PCR of
RNA from non-transfected Hep3B cells retrotranscribed with pcDNA reverse
primer, specific for TPO constructs. The use of pcDNA reverse primer
for cDNA synthesis allows the specific retrotranscription of transfected
constructs and not of the endogenous TPO mRNA. Water, control PCR
for reagents. RT +/–, presence/absence
of MMLV enzyme in the RT mixture.