The EF1α vector lacks LMO2 activation activity, whereas the Revgen vector activates LMO2 expression. (A) The provirus form of the Revgen and the EF1α vector was targeted to the first intron of the LMO2 gene locus in Jurkat T lymphocytes by Cre-mediated homologous recombination. The expression of LMO2 in the targeted Jurkat cell clones was analyzed by immunoblot. Clones 1 to 4 are 4 Jurkat clones targeted with the Revgen vector and clones 5 and 6 are 2 clones targeted with the EF1α vector. Human erythroid leukemic cell line K562 cells constitutively express high level of LMO2 and is used as positive control. Jurkat cell itself does not express detectable level of LMO2. LTR indicates Jurkat clone with a γ-retrovirus LTR-GFP cassette targeted into the first intron of the LMO2 gene in Jurkat cells; LTR(2F4), a subclone of the LTR clone that was culture for equivalent time as the Revgen and EF1α clones, showing the stability of LMO2 activation by the LTR enhancer; and ΔLTR, a Jurkat clone with the LTR-GFP cassette deleted by Cre-mediated recombination, showing that LMO2 expression is turned off. (B) Northern blot analysis of RNA preparations from the 4 Jurkat clones with targeted Revgen vector insertion. The full-length γc cDNA was used as probe. A single band of 1472 bp demonstrates that the γc transcript derived from the Revgen vector is indistinguishable from that of the endogenous γc allele of Jurkat cells. (C) Northern blot analysis of RNA preparations from the 2 Jurkat clones with targeted EF1α vector insertion. The full-length codon-optimized γc cDNA was used as probe. This probe cross-reacts with the endogenous γc RNA, which is expressed in Jurkat cells and is shown as a 1472-bp band. The RNA species transcribed from the EF1α vector is shown as another predicted main band of approximately 1.9 kb. A vertical line has been inserted between the EF1α clones and the Jurkat lane to indicate repositioned lanes of Jurkat and LTR from the same gel.