title: Recombineering BAC transgenes for protein tagging.
abstract: Protein tagging offers many advantages for proteomic and regulomic research, particularly due to the use of generic and highly sensitive methods that can be applied with reasonable throughput. 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. BAC (bacterial artificial chromosome) transgenes present a way to express a chosen protein at physiological levels with all regulatory elements in their native configurations, including cell cycle, alternative splicing and microRNA regulation. Recombineering has become the method of choice for modifying large constructs like BACs. Here, we present a method for protein tagging by recombineering BACs, transfecting cells and evaluating tagged protein expression.
authors.affiliation: Genomics, BioInnovationsZentrum, Technische Universitaet Dresden, Am Tatzberg 47, 01307 Dresden, Germany.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Ciotta
authors.forename: Helmut
authors.initials: H
authors.lastname: Hofemeister
authors.forename: Marcello
authors.initials: M
authors.lastname: Maresca
authors.forename: Jun
authors.initials: J
authors.lastname: Fu
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; D022202:Chromosomes, Artificial, Bacterial; D005818:Genetic Engineering / Q000379:methods; D049452:Green Fluorescent Proteins / Q000032:analysis; D051379:Mice; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014162:Transfection; D019076:Transgenes
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins