title: Recombineering, transfection, Western, IP and ChIP methods for protein tagging via gene targeting or BAC transgenesis.
abstract: 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).
authors.affiliation: Genomics, BioInnovationsZentrum, Technische Universitaet Dresden, Am Tatzberg 47, 01307 Dresden, Germany.
authors.forename: Helmut
authors.initials: H
authors.lastname: Hofemeister
authors.forename: Giovanni
authors.initials: G
authors.lastname: Ciotta
authors.forename: Jun
authors.initials: J
authors.lastname: Fu
authors.forename: Philipp Martin
authors.initials: PM
authors.lastname: Seibert
authors.forename: Alexander
authors.initials: A
authors.lastname: Schulz
authors.forename: Marcello
authors.initials: M
authors.lastname: Maresca
authors.forename: Mihail
authors.initials: M
authors.lastname: Sarov
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Anastassiadis
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D053595:Embryonic Stem Cells / Q000378:metabolism; D000939:Epitopes; D005838:Genotype; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D019076:Transgenes
chemical_list: D000939:Epitopes; D011993:Recombinant Fusion Proteins