Figure 2.
Mammalian 2′-phosphotransferase Trpt1 is required for Trl1-mediated rescue of RtcB cKO ES cells. A, representation of the mouse Trpt1 genomic locus, the targeting construct, and the targeted locus. After FLP recombinase–mediated recombination, the NeoR cassette is removed. In the Trpt1 knockout alleles, exons 2–6 are deleted. The Western blot shows the loss of Trpt1 expression in the generated Trpt1−/−, RtcB cKO ES cell line (T10D6). B, methylene blue staining shows the survival of RtcB cKO cells but not Trpt1, RtcB double KO cells with Trl1 expression in the presence of 4-OHT for 7 days. Western blots show that RtcB was depleted in Trl1 rescued cells and Trl1 was up-regulated after prolonged 4-OHT selection. C, methylene blue staining shows the genetic rescue of Trpt1, RtcB double KO T10D6 cells with Trl1 and Trpt1 after 4-OHT treatment. Western blotting verified the expression of Trpt1, RtcB, and Trl1 proteins.