Investigation of chromosome-mediated gene transfer using the HPRT region of the human X chromosome as a model.

Abstract

A panel of over 50 hybrid cells containing varying portions of the long arm of the human X chromosome have been obtained by chromosome-mediated gene transfer (CMGT) of human chromosomes to mouse cells deficient in HPRT. This panel is used to investigate the size and integrity of transfected human chromosome fragments and also to examine the effect of including a selectable DNA plasmid in the transfection mix. Chromosomal rearrangements are found to be generated in the chromosome transfer process, and the human X centromeric re#on is detected in the transfected cells at an unusually high frequency. Extensive lengths of X chromosome DNA are transferred intact, suggesting potential uses of CMGT in cloning large genes and loci for which only the chromosomal map position is known.[Key Words: Chromosome transfer; human gene mapping; X chromosome; centromere selection] Received November 6, 1986; revised version received and accepted January 5, 1987.Chromosome-mediated gene transfer (CMGT) can be used to construct hybrid cells that retain fragments of the selected donor chromosome (Mcbride and Ozer 1973}. Chromosome breakage is generally assumed to be random and the chromosome fragment size can vary from 50 kb up to a whole chromosome (Wullems et al. 1976;Olsen et al. 1981). The intermediate size of the transgenome produced by CMGT provides a method for linking and ordering genes over longer distances than the high-resolution mapping techniques of DNA-mediated gene transfer or cosmid cloning and at a more sensitive level than can be achieved by somatic cell hybrids or in situ hybridization. In addition, if fragmentation is random, the frequency with which loci are separated by CMGT could be used as a measure of genetic distance (for review, see Mcbride and Peterson 1980). Despite these advantages CMGT has been restricted in its applications. One major drawback has been the lack of suitable selective methods for isolating the cells containing the required chromosomal fragment. Until recently, only those regions of the genome encoding endogenous selectable loci were amenable to analysis, the best examples being the HPRT locus on the long arm of the human X chromosome (Miller and Ruddle 1978; and the TK locus on the long arm of human chromosome 17 (Klobutcher and Ruddle 1979).In previous experiments, new selection methods were used to extend the number of regions of the genome that could be subjected to analysis by CMGT (Pritchard and Goodfellow 1986). By cotransferring the selectable DNA plasmid, pSV2neo, with human chromosomes to mouse HPRT-cells, it was shown that the frequency of combined uptake of the HPRT chromosomal locus with pSV2neo was close to the frequency of uptake of HPRT alone. This observation demonstrated the feasibility of including an unlinked selectable marker in the transfection mix, followed by preselection for incorporation of that marker as an enrichment step in CMGT experiments. Furthermore, the fact that the neo gene and the HPRT chromosomal locus remained associated after a second chromosome tran...

Construction of a genetic map of human chromosome 17 by use of chromosome-mediated gene transfer.

“…We found CMGT to be particularly useful in the analysis of groups of closely linked markers, such as GALK, TKI, GHC, UMPH, and GAA, which could not easily be resolved by other means. Our results also confirm that (i) although substantial lengths of DNA may be transferred intact (8,9), interstitial deletions do frequently occur; (ii) multiple fragments of transfected chromatin can be found in the same clone; and (iii) there is also selection for centromeric sequences (8,9).…”

“…A total of 54 independent transfectant clones were isolated and analyzed by use of probes or isoenzymes for >20 loci located on chromosome 17. By combining the data from this chromosomemediated gene transfer transfectant panel, conventional somatic-cell hybrids containing well-defined breaks on chromosome 17, and in situ hybridization, we propose the following order for these loci: pter- (i) although substantial lengths of DNA may be transferred intact (8, 9), interstitial deletions do frequently occur; (ii) multiple fragments of transfected chromatin can be found in the same clone; and (iii) there is also selection for centromeric sequences (8,9).In addition, during the course of this study we obtained some transfectants that contained only the small regions of chromosome 17 particularly relevant to the study of APL and von Recklinghausen neurofibromatosis. …”

Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase chromosomes.

“…A large number of independent human DNA fragments (120) were screened in this study to obtain the two present in the CTPS gene, but, in retrospect, this difficulty could have been avoided by cloning fragments from a library containing larger human DNA inserts. It is known that rearrangements, particularly interstitial deletions, occur in the 'transgenomes' taken up by recipient cells (Porteous et al, 1986;Pritchard and Goodfellow, 1987). Our observations indicate that this process must be extensive in order to reduce the overlapping sequences in the primary transfectants to such a low level.…”

Chromosomes of older humans are more prone to aminopterine-induced breakage.

“…USA 86 (1989) convenient experimental tool to estimate physical distances between the corresponding loci (42). However, we now know that inborn or age-dependent variation in the individual proneness to induced chromosomal breakage can affect the accuracy of estimates of genetic distances obtained with the cell hybrid cotransfer method (5) or its modified version based on chromosome-mediated transfer (43). The persistence of a higher sensitivity of the old-age X chromosomes to aminopterine, even after their transfer in the cell hybrid genome, suggests that the aging process must have altered them in an irreversible manner and that, whatever their nature, these alterations are faithfully replicated at each cell division as "molecular scars" that have become imprinted in the chromosomes of aged individuals, thus becoming the preferential target sites for additional drug-induced chromosomal breakages.…”