Stable integration and expression in mouse cells of yeast artificial chromosomes harboring human genes.

Abstract

We have developed a way to fit yeast artificial chromosomes (YACs) with markers that permit the selection of stably transformed mamnalian cells, and have determined the fate and expression of such YACs containing the genes for human ribosomal RNA (rDNA) or glucose-6phosphate dehydrogenase (G6PD). The YACs in the yeast cell are "retrofitted" with selectable markers by homologous recombination with the URA3 gene of one vector arm. The DNA fragment introduced contains a LYS2 marker selective in yeast and a thymidine kinase (TK) marker selective in TK-deficient cells, bracketed by portions of the URA3 sequence that disrupt the endogenous gene during the recombination event. Analyses of transformed L-M TK-mouse cells showed that YACs containing rDNA or G6PD were incorporated in essentially intact form into the mammalian cell DNA. For G6PD, a single copy of the transfected YAC was found in each of two transformants analyzed and was fully expressed, producing the expected human isozyme as well as the heterodimer composed ofthe human gene product and the endogenous mouse gene product.To study gene expression, cloned fragments of DNA are often reintroduced into eukaryotic cells by transfection (1) or electroporation (2). Conventional vectors, however, have placed an upper limit on the size of DNA that can be studied, since inserts of no more than 40 kilobases (kb) can be accommodated, and it is now clear that many genes are larger than that. One way to circumvent this limitation has been provided by the development of yeast artificial chromosomes (YACs) as a cloning system, since DNA inserts in YACs can be as large as a megabase or more (3-7). We have designed a selective system to test the potential of transfected YACs for short-and long-term studies of gene expression.The test genes we have used are rDNA, encoding ribosomal RNA (rRNA), and the housekeeping gene G6PD, encoding glucose-6-phosphate dehydrogenase (G6PD). The number of natural variants detected for G6PD, now in excess of 300, is the largest known for any enzyme (8), providing unusually favorable material for studies of natural mutational variation and possible regulatory changes.Though the transcription units in the cases of rDNA and G6PD are well defined and are each contained in <20 kb, repeated searches have failed to recover the entire units in single A or cosmid clones. In contrast, YACs of 40-50 kb contain the entire genes (9, 10), and when a YAC containing G6PD was transfected into mammalian cells, the normal human isozyme was transiently expressed at easily detectable levels within 48 hr (11).Only a small number of cells take up DNA in standard assays with calcium phosphate-mediated transfections (1), and much of the DNA is either in the process of being degraded or is adventitiously stuck to cellular material. (14), which contains the URA3 gene, into pUC18 (15)]. The Stu I site is in the middle of the yeast URA3 gene of the plasmid, so that the net result of ligation is an 11-kb bacterial plasmid, TKLU2, containing the L YS2 and TK genes i...

Yeast artificial chromosomes spanning 8 megabases and 10-15 centimorgans of human cytogenetic band Xq26.

“…The sizes of restriction fragments generated by the rare cutters were also consistent with the map. (ii) In two cases where the analysis ofclones from the q arm of chromosome X has been pushed to the level of gene expression, copies of G6PD (26) and HPRT (27) Comparison ofPhysical and Genetic Maps. One way to align and orient contigs along the chromosome is provided by linkage probes that have been used to make genetic maps.…”

Modification of Bacterial Artificial Chromosome Clones Using Cre Recombinase: Introduction of Selectable Markers for Expression in Eukaryotic Cells

“…All 10 retrofitted clones show identical bands, whereas the parental clone has three differences. (Eliceiri et al 1991). Subsequent retrofitting strategies used Tn10-mediated insertion of neo into P1 clones .…”

Plant Molecular Biology — A Laboratory Manual

“…1992), the most significant successes have co me from mamma li an genome research. 1992;Bellann-Chantelot et al 1992) and the relative ease of introducing Y AC clones into mammal cells by using appropriate selectable markers in the vector, enabling the functional analysis of large gene units (Elicieri et al 1991;Jakobovits et al 1993;Schedl et al 1993). 1992;Bellann-Chantelot et al 1992) and the relative ease of introducing Y AC clones into mammal cells by using appropriate selectable markers in the vector, enabling the functional analysis of large gene units (Elicieri et al 1991;Jakobovits et al 1993;Schedl et al 1993).…”