期刊
METHODS
卷 53, 期 4, 页码 437-452出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2010.12.026
关键词
Recombineering; BAC transgenesis; Gene targeting; Protein tagging; Native affinity purification; Western blotting; Chromatin immunoprecipitation; Genotyping by long range PCR
资金
- EU
- BundesMinisterium fuer Bildung und Forschung
Protein tagging offers many advantages for proteomic and regulomic research. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. Physiological expression of tagged proteins can be achieved by gene targeting to knock-in the protein tag or by BAC transgenesis. BAC transgenes usually retain the native gene architecture including all cis-regulatory elements as well as the exon-intron configurations. Consequently most BAC transgenes are authentically regulated (e.g. by transcription factors, cell cycle, miRNA) and can be alternatively spliced. Recombineering has become the method of choice for generating targeting constructs or modifying BACs. Here we present methods with detailed protocols for protein tagging by recombineering for BAC transgenesis and/or gene targeting, including the evaluation of tagged protein expression, the retrieval of associated protein complexes for mass spectrometry and the use of the tags in ChIP (chromatin immunoprecipitation). (C) 2011 Elsevier Inc. All rights reserved.
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