PCR on isolated tissues.

(A) Presence of Neo^R gene in hematopoietic organs from sheep transplanted in utero with retrovirally marked stromal cells. Following sacrifice, single cell suspensions of liver, spleen, thymus, and BM were prepared as detailed in “Materials and methods,” and the DNA obtained from these cells was then subjected to PCR analysis with primers specific for the vector-encodedNeo^R gene. The reagent control consisted of all of the constituents of the PCR reaction mixture except template DNA (lane 1 from the left). The negative control (−) DNA was isolated from the PB mononuclear cells from a normal control ram (lane 2). The positive control consisted of the plasmid pUC18Neo diluted in normal sheep DNA to a concentration of 1% (lane 11). The remainder of the samples consisted of DNA extracted from the organs (as labeled in the figure) of 2 different transplanted sheep. For each organ there are 2 samples; the sample on the left is from the time point of 2 weeks after transplant, and the sample on the right from 6 weeks after transplant. (B) SCF RT-PCR on marrow stromal cells. To evaluate whether the transplanted stromal cells were expressing mRNA for hematopoietic growth factors BM stromas grown from 5 sheep transplanted with human stroma cells alone were harvested and RNA was isolated. This RNA was then reverse transcribed into cDNA and used as a template for SCF-specific PCR. The reagent control consisted of all of the reaction constituents except template DNA, and the negative control consisted of RNA isolated from the BM mononuclear cells of a normal control sheep (lane 1). The positive control was RNA isolated from human BM mononuclear cells (lane 8). Lanes 2 to 6 consisted of RNA extracted from stromal layers cultured from BM of 5 different sheep injected with human stromal cells alone. In all but 1 animal (lane 5) we were able to detect mRNA for SCF. We were able, however, to amplify a fragment of β-actin message from this sample demonstrating the presence of intact RNA.