In the presence of biologically relevant concentrations of magnesium and guanosine nucleoside, some introns, called group I introns, are capable of self-splicing: excising themselves from the flanking RNA, and religating the RNA exons to form a functional RNA.
pCZ486 contains a deletion only in the drsh-1 coding region and was constructed by digesting pCZ483 with SphI and religating.
Thus, mutation of the 4BS element was achieved by linearizing pmin0−GFP with Tfi I, using mung bean exonuclease to remove ssDNA overhangs, and religating with T4 DNA ligase.
The one adjacent to the EF1-NLS-βGal coding region was eliminated by linearizing the plasmid through a partial digest and blunt ending the vector and religating the blunt ends.
The pSico-Rac1 lentiviral vector was produced by digesting the pSico construct with HpaI-XhoI restriction enzymes (Promega), inserting shRNA oligonucleotides to target the mouse Rac1 gene and religating using T4 DNA ligase (Promega).
Mechanistically, Topo II functions to alter DNA topology by catalyzing the ATP-dependent passage of one DNA double helix through another by breaking and religating one of the DNA strands while transporting the second [26], [29], [30], [31].
Empty vector control was obtained by cleaving the IFI16 insert out of CMV-SPORT6 using NotI/ SalI and religating the vector.
The DNA encoding eGFP was removed from pAct5eGFP by cutting with BamHI and XbaI, blunting using Klenow polymerase and religating to yield pAct5c.
The BX donor was constructed by digesting the 7.5-kb donor with XbaI and BspDI, then blunting and religating as above, creating a 1232-bp deletion beginning at the target site.
The SA donor was constructed by digesting the 7.5-kb donor with AflII and SgrAI and blunting and religating as above, creating a 1307-bp deletion beginning 699 bp upstream and ending 608 bp downstream of the target site.
