Fig. 4.
Retrohoming of Ll.LtrB Lin RNPs in D. melanogaster embryos. Retrohoming assays using Lar and GG-Lin RNPs were done in D. melanogaster precellular blastoderm embryos, as described in Fig. 1 and Materials and Methods. After incubating the embryos for 30 min at 37 °C, nucleic acids were extracted and either transformed into E. coli HMS174(DE3) for plating assays and colony PCR or used directly for PCR analysis of integration products, as described in Figs. 2 and 3 for X. laevis oocyte assays. (A) E. coli colony PCR of 5′ integration junctions in randomly selected TetR + AmpR colonies obtained after retrohoming of GG-Lin RNPs. (B) Direct PCR analysis of 5′ and 3′ integration junctions using primers P1 and P2 for the 5′ junction and primers P3 and P4 for the 3′ junction (Materials and Methods). (C) Sequences of 5′ junctions from colony PCR products. (D) Sequences of 5′ junctions in PCR products amplified from extracted DNA without transformation into E. coli. Intron and exon sequences are depicted as in Fig. 2.