从通过酵母中的TA克隆进行选择性全长基因分离到其在人类细胞中从HAC载体表达。

From selective full-length genes isolation by TAR cloning in yeast to their expression from HAC vectors in human cells.

作者信息

Kouprina Natalay, Lee Nicholas C O, Kononenko Artem V, Samoshkin Alexander, Larionov Vladimir

机构信息

Laboratory of Molecular Pharmacology, National Cancer Institute (NCI, NIH), Bldg 37 Room 4 E28, 37 Covent Drive, Bethesda, MD, 20892-4255, USA,

出版信息

Methods Mol Biol. 2015;1227:3-26. doi: 10.1007/978-1-4939-1652-8_1.

DOI:10.1007/978-1-4939-1652-8_1

PMID:25239739

Abstract

Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed.

摘要

转化相关重组（TAR）克隆技术可在酵母中作为大型环状酵母人工染色体（YAC）选择性分离全长基因和基因组位点。该方法在结构和功能基因组学、长程单倍型分析、染色体重排特征研究及进化研究等方面有广泛应用。本文描述了通过TAR进行基因分离的基本方案，以及使用改造载体将TAR分离物转化为细菌人工染色体（BAC）的方法。该改造载体包含一个3'HPRT-loxP盒，以便随后将基因加载到基于HAC（人类人工染色体）的基因递送载体的独特loxP位点。讨论了将TAR基因克隆技术与HAC基因递送系统相结合用于基因表达研究的益处。