Fig. 3.
Retrotransposition activity analysis of pig L1. a Schematics of vectors used for retrotransposition assays. hL1 and mhL1 were used as positive and negative control, respectively. The pL1 vector contains 5′UTR, ORF1, IGR, ORF2, and 3′UTR of L1 cloned from the pig genome (L1D1 coordinate). The pL1-CMV is the same as pL1, but the 5′UTR of pig L1 was replaced with the CMV promoter. The phL1 is a chimeric vector derived by the CMV promoter, the two ORFs and 3′UTR were from pig, and the IGR was from human L1 (99-PUR-RPS-pBlaster1). All the vectors contain two selective cassettes (mBlast and Puro) for two-round selections. The mBlast cassette contains an inverted blasticidin resistance gene (black box) disrupted by a self-splicing intron [49–51]. The introns will only splice out from a transcript generated by the L1 or CMV promoter. The spliced RNA is reverse-transcribed, followed by integration of the cDNA into the genome. The new insert contains a functional Blast gene. Blasticidin resistance will be obtained only if retrotransposition occurs. b and c Number of clones formed after puromycin and blasticidin selection. BlastR foci were fixed to flasks and stained with Giemsa for visualization. Bars represent the mean blasticidin resistant colonies ± standard deviation, shown as error bars for each construct