Vol. 116, No. 3, 1983 November 15, 1983 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 959-965 LACK OF SITE SPECIFIC RECOMBINATION OF EXOGENOUS DNA IN MOUSE L CELLS Patricia E. Berg + , Ann Henderson*, Sally Ripley*, Jian-Kang Yu +, and W. French Anderson + +Laboratory of Molecular Hematology National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland 20205 *Department of Human Genetics and Development College of Physicians and Surgeons of Columbia University New York, New York 10032 Received September 27, 1983 SUMMARY: Plasmids were constructed containing the HSV thymidine kinase 9ene and two copies of X. borealis 5S rDNA. Mouse L TK- cells were transformed with these DNAs, with selection for the TK + gene. Transformed cells were then analyzed by Southern blot hybridization and hybridization in situ to determine whether integration of the exogenous DNA occurred at regions of ~hromosomal homology i.e., at the 5S rDNA regions. Four cell lines were analyzed by Southern blots. Differences in restriction endonuclease specificity strongly suggested that integration was at a different site in each cell line. Two c e l l lines were further analyzed by hybridization in situ; each showed a single integration site, both different from each other and different from the mouse L cell 5S rDNA sites. Therefore, the presence of two copies of the 5S rDNA gene in the DNA introduced by gene transfer and approximately 300-350 copies of the mouse 5S rDNA gene was not sufficient in these experiments to produce homologous integration into a specific site. Homologous recombination, i.e., recombination between DNA sequences sharing homology, frequently occurs in procaryotic organisms and in lower eucaryotes such as yeast (I). It has been assumed that a comparable system might exist in mammalian c e l l s . However, although homologous recombination does occur between different incoming DNA molecules (2-7), no homologous recombination between newly introduced DNA and chromosomal DNA has yet been identified (8). Plasmid or SV40 DNA introduced into mammalian cells appears to integrate randomly into the chromosome (2,3,8-11) since there is no apparent relationship between the site of integration and the normal chromosomal DNA sequence or any morphological feature of the chromosome in mammalian cells. There has not been reported thus far a specific attempt to site-direct the integration of an exogenous gene. 0006-291X/83 $I .50 Copyright © 1983 by Academ~ Press, In~ 959 AH righ~ of reproduct~n m any form reserved.