(A) RT-PCR detection of MDR1 expression by multifactor HPP-CFC. Single murine HPP-CFC colonies were picked and assayed for expression of the transferred human MDR1 gene using the RT-PCR assay as described in Materials and Methods. The first set of three lanes is from a 6-factor HPP-CFC (HPP#3) transduced with the MDR1 vector. This colony, as well as HPP#2, HPP#1, HPP#6, and HPP#4, all contain the RT-PCR fragment corresponding to the mRNA from the transferred human MDR1 vector sequence. Each lane represents RNA equivalent to $112$ of a colony. Samples are run in duplicate with a no-primer (no 1°) control. No-template negative control reactions are interspersed among the assay samples. The last sets are NIH3T3 RNA (negative control, murine cell line), MDR18.1 RNA (positive control, MDR1 retroviral producer line), and the tRNA/agar control for background (negative control for nonspecific signals and the carrier in the RNA isolation protocol). (B) Colony RT-PCR assay controls. This panel shows the results of the control templates routinely run during the RT-PCR assay for MDR1 expression. Duplicate control templates are amplified concurrently with test samples using the same reagents and conditions; a no-primer (no 1°) control is also included for each. Control templates (from left to right) are (1) tRNA carrier, which is present in the RNA extractions: This gives no RT-PCR background when SSRTII is used for reverse transcription; (2) agar from between colonies: Neither the agar nor any extraneous material from the transduction or plating generates RT-PCR background; (3) MDR18.1 RNA from the producer cell line: 1 μg of total cellular RNA in the RT step is included as a positive control; (4) NIH3T3 cell line RNA (or murine bone marrow RNA): 1 μg of total cellular RNA in the RT step is included as a negative control (this is done to detect any cross reaction with any of the MDR isoforms present in mice); (5) No-template reaction: This is a standard PCR control for each set of primers used; and (6) a nontransduced multifactor murine HPP-CFC colony: This is processed identically to the experimental samples so that each lane represents $112$ of a colony.