Recombination in mouse L cells between DNA introduced into cells and homologous chromosomal sequences.

Abstract

In this paper, we show that DNA added to mouse L cells by the calcium phosphate method can be inserted into the genome of those cells by homologous recombination. The insertion event is detected because it reconstructs a functional thymidine kinase (tk) gene from two defective genes that share 320 base pairs of homology. One of the genes is missing its 5' portion (tkA5') and is in the cell's chromosome, and the other is missing its 3' portion (tkA3') and is in the introduced DNA. 40 (6, 7, 9) indicates that homologous recombination between extrachromosomal DNA and chromosomal DNA is possible in mammalian cells. The use of viruses, however, to analyze this sort of recombination severely limits further studies. In particular, it is difficult to quantitate the process by measuring the yield of recombinant virus and, without a method of isolating and analyzing both partners in a recombination event, one can say little about mechanism. Furthermore, it probably will be necessary to develop viral-free gene transfer techniques to make use of recombination events that target DNA into homologous chromosomal loci.

Extrachromosomal and chromosomal gene conversion in mammalian cells.

“…Since site-specific integration of genes appears to be extremely rare (8,16,17), it would be of interest to determine whether genetic mutations can be corrected by gene conversion from segments of DNA introduced artificially elsewhere into the genome. We are also exploring whether gene conversion can be used as a mechanism to exchange genetic information between replicating extrachromosomal molecules and the chromosomes of mammalian cells.…”

Theoretical mechanisms in targeted and random integration of transgene DNA

“…The reasons for this are not known, but the effect seems to exist. For example, separate studies were conducted by Lin et al [25] and Thomas et al [42] both involving gene targeting of artificially introduced defective genes in mouse fibroblasts. Lin et al reported a ratio of random integration to gene targeting of 100 000:1 whereas Thomas et al reported a ratio of 100:1.…”

DNA looping by Ku and the DNA-dependent protein kinase

“…In yeast, DSB repair occurs largely by homologous recombination (4)(5)(6)(7). Although capable of homologous recombination, higher eukaryotic species repair DSBs primarily by a process of nonhomologous or illegitimate recombination (8)(9)(10)(11)(12)(13).…”