Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts.

Abstract

We have examined the ability of singlestranded DNA to participate in homologous recombination reactions in mammalian cells and nuclear extracts derived from them. We have inserted a fragment of the neo gene into the single-stranded DNA phage vector M13 mphl. The neo fragment was derived from a deletion derivative of the prokaryotic-eukaryotic shuttle vector pSV2neo. The resulting singlestranded DNA was mixed with a double-stranded deletion derivative of pSV2neo and tested for recombination in human cells, monkey cells, and nuclear extracts obtained from human cells. We were able to obtain recombinant molecules containing wild-type neo genes in all three systems. Examination of the products of recombination indicated that they resulted from correction of the deletion in the double-stranded DNA substrate. We were unable to detect any extensive conversion of single-stranded DNA into its double-stranded counterpart before it participated in the recombination reaction. We have also tested the ability of single-stranded DNA to yield transfectants. When a single-stranded DNA derivative of the herpes simplex virus thymidine kinase (TK) gene was introduced into mouse L-M(TK-) cells, we were able to obtain TKV colonies.From these results, we conclude that single-stranded DNA can participate in transfection as well as homologous recombination reactions in mammalian cells.Integration of exogenous DNA into mammalian chromosomes by homologous recombination would be the preferred strategy for gene replacement therapy and in vivo gene modification. Mammalian somatic cells have been shown to contain all of the enzymes required to mediate recombination between homologous viral or plasmid sequences cotransfected into cells or between chromosomal sequences and their homologous counterparts (1-6). The exact nature of the proteins involved and the mechanism by which homologous recombination occurs in mammalian cells is not understood.We have developed a set of three systems to study the genetic and biochemical aspects of homologous recombination in mammalian cells (7-9). All of these involve the use of two derivatives of the prokaryotic-eukaryotic shuttle vector pSV2neo, a plasmid containing the bacterial gene for aminoglycoside 3'-phosphotransferase, which can be expressed in bacterial as well as in mammalian cells, resulting in neomycin or kanamycin resistance in bacteria and G418 resistance in mammalian cells. It also has an ampicillinresistance (AmpR) gene. Each of the two recombination substrates is a nonoverlapping, nonreverting deletion derivative of pSV2neo. The two deletion derivatives are referred to as pSV2neo DL and pSV2neo DR (7). In the first system, homologous recombination is studied by cotransfecting the two deletion plasmids into mammalian cells and selecting the cells for the expression of the normal neo gene, which can be generated by homologous recombination (7). The second method, somewhat analogous to studying bacteriophage recombination, involves introducing these substrates into monkey COS cells (10) ...

Homologous recombination enhancement conferred by the Z-DNA motif d(TG)30 is abrogated by simian virus 40 T antigen binding to adjacent DNA sequences.

“…To determine whether recombination was occurring in COS-1 cells, we cointroduced DL and DR molecules into the cells and calculated the Kanr/Ampr frequency of low-Mw DNA isolated 48 h later. Cotransfection of DL with DR resulted in a 7.5 x 10-frequency of Kanr/Ampr colonies (Table 1), consistent with previous studies (1,33). Because both of the substrate molecules contain deletions within the neo gene, the only way that Kanr colonies may be generated is via a homologous recombination event.…”

“…Large numbers of extrachromosomal product molecules may be isolated, circumventing some of the difficulties associated with product analysis from nonreplicating systems. Previous studies indicate that recombination between replicating substrates occurs by the same types of mechanisms as in nonreplicating systems (1,33,35). In addition, it has been shown that double-strand breaks, which stimulate recombination in a variety of systems (reviewed in reference 43), are recombinogenic within replicating systems as well (1).…”

Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells

“…Further, we confirmed the differential activity of HR and NHEJ pathways in mESC and fibroblasts by in vitro functional assays [39][40][41]. Recombination reactions using nuclear protein extracts prepared from exponentially growing cells showed a 3-fold increase in HR activity in mESC compared to primary fibroblasts (P value <0.001; Fig.…”

“…HR catalyzed by cell-free extracts was performed essentially as described previously [39], using a plasmid substrate pair: SalI linearized pSV2neoDL and single-stranded circular ssneoDR, which were kindly provided by Dr. Colin Campbell, University of Minnesota. Briefly, 500 ng of each of these substrates were incubated with nuclear protein extracts prepared from ear fibroblasts or ESC at 378C for 2 h. Purified plasmid DNA was used to electroporate the recombinationdefective Escherichia coli strain DH10B using a Bio-rad Gene Pulser XcellÔ.…”

Is it possible to improve homologous recombination in Chlamydomonas reinhardtii?

“…Other studies scrutinized the potential of ssDNA to increase the rate of HR in gene targeting experiments (Rauth et al 1986;Simon & Moore 1987;Baur et al 1990;Bilang et al 1992;Zorin et al 2005) and since single-stranded DNA plays a central role in all proposed recombination models, the use of single-stranded targeting vectors is a promising approach for improving HR. Taken together, most of these experiments demonstrated an increased rate of HR.…”