Transfer of the human HPRT and GART genes from yeast to mammalian cells by microinjection of YAC DNA

Abstract

DNA of two yeast artificial chromosomes (YACs) containing selectable human genes was transferred by microinjection to rodent cells in tissue culture. The human hypoxanthine phosphoribosyltransferase (HPRT) gene, spanning 45 kb, is contained on the 660-kb YAC yHPRT as described elsewhere. The human phosphoribosylglycinamide formyltransferase (GART) gene, spanning approximately 40 kb, is contained on the 590-kb YAC yGART2 as described previously. YAC DNA was isolated from pulsed-field gels and microinjected into mammalian cells in which the human HPRT and GART genes can be selected. The cell lines that were selected contain the entire human genes. Some of the cell lines contain multiple copies of the genes integrated at the same chromosomal position. The YAC yGART2 could not be purified away from natural yeast chromosomes of similar size, and the cell lines into which the human GART gene was introduced contain variable amounts of yeast DNA in addition to the human DNA.

Genetic transfer and expression of reconstructed yeast artificial chromosomes containing normal and translocated BCL2 proto-oncogenes.

“…YACs ranging in size from 35 to 680 kb have been introduced stably into mammalian cells and mouse genomes. Protoplast fusion, microinjection, and lipofection were used to introduce larger DNA fragments (> 150 kb) (15,16,24,25,31,32,35), whereas calcium phosphate precipitation (11,12) was used to introduce smaller fragments. The percentage of transfected cells containing the entire YAC insert (± vector arm sequences) ranged from 10 to 47% (23).…”

Transgenic Animals with Various YAC Constructs: Preparation of 400 kb YAC DNA for Microinjection

“…The YAC DNA isolated by PFGE is not of sufficient concentration for microinjection. It is difficult to concentrate YAC DNA by conventional methods such as ethanol precipitation due to mechanical shearing [34]. Schedl et al [9] used the method of YAC copy number amplification [17] to make DNA plugs which have concentrated YAC DNA.…”

Yeasts

“…They accomodate extremely large DNA fragments (up to 2 Mbp;COFFEY et al, 1992) and are used for mapping of mammalian DNA as well as isolation and propagation of intron-containing genes encoded by large DNA fragments (ANAND, 1992). In several cases, expression of genes carried on a YAC vector in mammalian cells has been reported (e.g., GNIRKE and HUXLEY, 1991;. Finally, if high copy numbers are needed for maximal expression, YEp vectors carrying a 2 pm origin of replication plus the STB region have been largely employed (VOLKERT et al, 1989;CHRISTIANSON et al, 1992).…”