Extrachromosomal Maintenance and Amplification of Yeast Artificial Chromosome DNA in Mouse Cells

Highly selective isolation of human DNAs from rodent–human hybrid cells as circular yeast artificial chromosomes by transformation-associated recombination cloning

“…Although there have been several reports on the cloning of DNA as large circular molecules in yeast (19,20), their stability has not been well characterized. We have established that TARcloned circular YACs exhibit structural and segregational stabil- Genetics: Larionov et al Proc.…”

Establishment of an oriP/EBNA1-based episomal vector transcribing human genomic β-globin in cultured murine fibroblasts

“…The blot was probed with an AlfIII-digested pBH140 vector that was used to generate the 200-kb pBH148. 10 All the A9pBH148 episomes iso- lated from any culture to week 12 (lanes [5][6][7][8][9][10][11][12][13][14] migrated at approximately the same position as the episome from the positive control DRpBH148 (lane 1). A DNA ladder, from the ethidium bromide-stained gel, is superimposed on the Southern blot to illustrate the distances between the intact 200-kb pBH148 circular episome, any sheared linear pBH148, and genomic fragments.…”

“…YACs, with up to 1500-kb of human DNA, replicated as episomes in mouse cells following yeast spheroplast fusion, but were unstable in the absence of selective pressure. 13,14,40,41 Although these YACs contained large human DNA fragments (complete with replication origins) they were devoid of any segregation apparatus. In other experiments, use of microcell fusion demonstrated that as little as 100-kb of human DNA could be maintained episomally in EBNA1-expressing mouse cells in low copy number, presumably by EBNA1 protein binding to the oriP on these vectors.…”

“…[2][3][4][5] Self-replicating viral-based vectors are capable of longterm episomal persistence in mammalian cells. 6,7 These vectors contain components derived from (1) EpsteinBarr virus (EBV; oriP/EBNA1 viral elements); [8][9][10] (2) bovine papilloma virus (BPV-1; ori elements); 11,12 (3) genomic components from either yeast (YACs) or human chromosomes (HACs); [13][14][15][16] and (4) chimeric viral/genomic systems either engineered from the oriP/EBNA1-based pLIB or from mammalian artificial episomal chromosomes (MAECs). [17][18][19] The advantages and limitations of these various viral vector-based systems in biological research are discussed elsewhere.…”

Complementation of the beige mutation in cultured cells by episomally replicating murine yeast artificial chromosomes.

“…2). This is probably due to the hybridization of (14), possibly because they contain an origin of replication (15). Some of our YAC transformants exist extrachromosomally and are amplified in high G418 concentrations, whereas others at these same drug concentrations are not amplified.…”