Fig. 4.

Integration of ancML into CHO cell DNA. a Example of a genome walker experiment to determine the 3′ flanking sequence of ancML integration events in 15 single cell clones that became resistant to G418 following transfection with ancML. Nested PCR reactions were done using EcoRV digested, adapter ligated, gDNA from single G418-resistat cell clones. Forward and reverse primers were designed to anneal to the R region of the 3′LTR and to the adaptor sequence, respectively. M: molecular weight ladder. u: CHO DNA without an integrated ancML insertion. b Top: the sequence of an integration site with both 5′ and 3′ flanking CHO gDNA. The five-nucleotide target site duplication is indicated in yellow. Bottom: Sequences of 26 ancML integration sites in the CHO genome. Sequences of the ancML U5-PBS region as well as the Leucine (TAA) tRNA sequence are included at the bottom of the diagram. Sequence from the U5 region of the 3′ ancML LTR is indicated in blue. The 5nt linker sequence is indicated in black. The PBS sequence is indicated in purple. CHO genomic sequences are indicted in bold. Dotted lines indicate correspondence of each sequenced 3′ integration junction to the integration site at the top. c Enumeration of G418 resistant colonies of CHO cells transfected with plasmids expressing a neo gene (NEO), L1.3, ancML and an ancML construct with a deletion of the 5nt linker sequence between the 5′ LTR and the PBS (ancML ∆GAAGT). Data are mean ± SD from 3 independent experiments. d Distribution of ancML integration sites in genic, intergenic, or repeat regions relative to matched random controls. The measured value indicates the percentage of ancML integration sites in each population divided by that of the matched random controls (each integration site was matched to three random genomic sequences equidistant to the EcoRV site where the adaptor was ligated). The horizontal dashed line indicates no difference between the frequencies of ancML integration sites in each population compared to the matched controls