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

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

Nature protocols (2013-07-23)
Liangping Li, Thomas Blankenstein
要旨

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-α (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.

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製品内容

Sigma-Aldrich
エチレンジアミン四酢酸 二ナトリウム塩 二水和物, suitable for electrophoresis, for molecular biology, 99.0-101.0% (titration)
Sigma-Aldrich
シクロヘキシミド, from microbial, ≥94% (TLC)
Roche
抗-ジゴキシゲニン-AP、Fabフラグメント, from sheep
Sigma-Aldrich
酵母ニトロゲンベース アミノ酸不含, Yeast classification medium used for selecting yeasts based on amino acid and carbohydrate requirements
Sigma-Aldrich
マイトマイシンC Streptomyces caespitosus由来, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
ESGRO®リコンビナントマウスLIFタンパク質, ESGRO Leukemia Inhibitory Factor (LIF) supplement for mouse ES cell culture. Each vial contains 10^7 units/ml.
Roche
PCR DIGプローブ合成キット, sufficient for 25 reaction (50 μL final reaction volume)