Expression of the tk gene inserted into the α_IIb locus.

(A) Results of Northern blot analysis of BM RNA prepared from wild-type mice (+/+), heterozygous (+/−), and homozygous tk-knocked mice (−/−) are represented. Each lane contains 30 μg total RNA. RNA integrity was determined by gel electrophoresis and visual examination of ethidium bromide-stained ribosomal bands. RNA samples were transferred to Hybond N+ nylon membrane (Amersham) and hybridized with α_IIb (left) or tk (right) probes. The distortion of the tk band might result because tk transcripts comigrate with the 18S ribosomal RNA. (B) Standard curves for α_IIb and tk transcripts. Serial dilutions of BM cDNA were used. 10 μL cDNA solution, corresponding arbitrarily to 2000 U, was used as the highest value. Cτ values are plotted against the relative concentration of serial-diluted cDNA. A strong linear relationship between the Cτ (the cycle threshold value predictive of the quantity of input target) and the log of the starting concentration of the cDNA is demonstrated (R² ≥ 0.98). (C) Relative expression levels obtained after semiquantitative RT-PCR of RNA prepared from a variety of tissues of heterozygous mice. Duplicates for each sample were performed, but the data for only one is shown here. A negative PCR control without reverse transcription was performed to exclude PCR amplification of contaminating genomic DNA. Dark and dashed boxes correspond to α_IIb and tk expression, respectively. Lane 1, BM; lane 2, spleen; lane 3, liver; lane 4, lung; lane 5, brain; lane 6, testes; lane 7, heart; lane 8, thymus; lane 9, kidney.