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. 2001 Feb 15;29(4):886–894. doi: 10.1093/nar/29.4.886

Figure 1.

Figure 1

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.