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Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast-embryonic stem cell fusion

Item Type:Article
Title:Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast-embryonic stem cell fusion
Creators Name:Li, L. and Blankenstein, T.
Abstract:Introducing human genes into mice offers the opportunity to analyze their in vivo function or to obtain therapeutic molecules. For proper gene regulation, or in case of multigene families, megabase (Mb)-sized DNA fragments often have to be used. Yeast artificial chromosome (YAC)-mediated transgenesis is irreplaceable for this purpose, because alternative methods such as the use of bacterial artificial chromosomes (BACs) cannot introduce DNA fragments larger than 500 kb into the mouse germ line. However, YAC libraries often contain only partial gene loci. Time-consuming reconstruction of YACs, genetic instability and the difficulty in obtaining intact YAC DNA above a certain size impede the generation of humanized mice. Here we describe how to reconstruct YACs containing Mb-sized human DNA, such as the T cell receptor-{alpha} (TRA) gene locus, thus facilitating the introduction of large DNA fragments into the mouse germ line. Fusion of YAC-containing yeast and embryonic stem (ES) cells avoids the need for YAC DNA purification. These ES cells are then used to stably introduce the functional TRA gene locus into the mouse germ line. The protocol takes ∼1 year to complete, from reconstruction of the entire TRA gene locus from YACs containing partial but overlapping TRA regions to germline transmission of the YAC.
Keywords:Yeast Artificial Chromosomes, Embryonic Stem Cells, Gene Transfer Techniques, Genetic Engineering, Genetic Markers, Homologous Recombination, Spheroplasts, Animals, Mice
Source:Nature Protocols
ISSN:1754-2189
Publisher:Nature Publishing Group (U.K.)
Volume:8
Number:8
Page Range:1567-1582
Date:August 2013
Official Publication:https://doi.org/10.1038/nprot.2013.093
PubMed:View item in PubMed

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