Recombineering BAC transgenes for protein tagging.
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.
Ciotta
Giovanni
G
Genomics, BioInnovationsZentrum, Technische Universitaet Dresden, Am Tatzberg 47, 01307 Dresden, Germany.
Hofemeister
Helmut
H
Maresca
Marcello
M
Fu
Jun
J
Sarov
Mihail
M
Anastassiadis
Konstantinos
K
Stewart
A Francis
AF
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
D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins