Two teams of researchers report in today's issue of Nature thatthey have successfully introduced large segments of humanDNA into mice using yeast artificial chromosomes, or YACs.

The DNA remains intact, its presence doesn't interfere with thedevelopment of the engineered embryonic stem cells intoadults, and it is passed through the germline. It now becomesfeasible -- at least in theory -- to study precisely the regulationand expression of human immunoglobulin genes, for instance,or to study human genetic disease genes in laboratory mice, oreven to engineer the animals to crank out quantities of humanmonoclonal antibodies for therapeutic uses.

Aya Jakobovits and her colleagues at Cell Genesys Inc.(NASDAQ:CEGE) of Foster City, Calif., introduced large segmentsof human DNA into murine embryonic stem (ES) cells by fusionwith yeast spheroplasts. The spheroplasts carried a 670-kilobase YAC containing the human hypoxanthinephosphoribosyltransferase (HPRT) gene. The researchers fusedthe spheroplasts with an HPRT-deficient ES cell line, and simplyselected for clones expressing the HPRT gene. Analysesindicated that large parts of the yeast genome co-integratedinto the ES cells along with the human sequences, and that theintegrated construct retained its structural integrity.

The engineered stem cells were able to differentiate in vitro and in vivo : Chimeric mice transmitted theintegrated foreign DNA fragments to their offspring. "Wedetected the expression of the human HPRT gene in spleen,liver, brain, heart and kidney, demonstrating that thetransmitted YAC DNA retained its function with fidelity," theauthors reported. "Our findings open the way for the use ofmegabase-sized loci for genetic complementation studies ofrecessive genetic disorders as well as for the generation ofnovel mouse strains," they concluded.

The second group of researchers hails from the German CancerResearch Center in Heidelberg. Andreas Schendl and hiscolleagues also used YACs to transfer large segments of DNAinto mouse embryos. In this case, the researchers transferred a250-kilobase YAC containing the mouse tyrosinase gene (the 80kilobase coding region) into mice by pronuclear injection intofertilized oocytes. The tyrosinase marker gene could bedetected by the appearance of pigment in the eye and skin ofnewborn mice expressing the albino phenotype.

Once again, "the YAC was inserted into the mouse genomewithout major rearrangements. ... Expression from thetransgene reached levels comparable to that of the endogenousgene and showed copy number dependence and positionindependence," the authors wrote.

"We envisage that YACs covering a locus and sufficient flankingsequences will be of great use for transgenic experimentswhere correct expression is essential. This will benefit theanalysis of large gene clusters in their natural context...and offar upstream regulatory elements," the researchers concluded.

In his analysis of and comments on the two papers, MarioCapecchi (the "inventor" of homologous recombination, apowerful gene targeting technique for transgenesis and aprofessor at the Howard Hughes Medical Institute, Universityof Utah School of Medicine) said, "The potential now exists forresearchers to use the YAC transfer technology to define thelocation of a gene on a large DNA fragment by introducing YACscontaining genomic fragments into mutant mice and scoring forthe rescue of the mutant phenotype."

But there are other applications where this technology will beof "enormous benefit," he said, especially in the "molecularanalysis of mammalian biology."

"The technology has far-reaching implications," commentedJordan Gutterman, chief of the department of immunology andbiological therapy at M.D. Anderson Hospital and TumorInstitute and a scientific adviser to Cell Genesys. "Now large locican be studied. There are many potential applications forunderstanding recessive genetic disorders by making mousemodels. ... It brings us much closer to a model for humandisease," he told BioWorld. For Gutterman, "human YACs" rankas one of the eight or 10 major genetic technologies to bedeveloped over the last 30 years.

-- Jennifer Van Brunt Senior Editor

(c) 1997 American Health Consultants. All rights reserved.

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