BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.
The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.
Poser
Ina
I
Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
Sarov
Mihail
M
Hutchins
James R A
JR
Hériché
Jean-Karim
JK
Toyoda
Yusuke
Y
Pozniakovsky
Andrei
A
Weigl
Daniela
D
Nitzsche
Anja
A
Hegemann
Björn
B
Bird
Alexander W
AW
Pelletier
Laurence
L
Kittler
Ralf
R
Hua
Sujun
S
Naumann
Ronald
R
Augsburg
Martina
M
Sykora
Martina M
MM
Hofemeister
Helmut
H
Zhang
Youming
Y
Nasmyth
Kim
K
White
Kevin P
KP
Dietzel
Steffen
S
Mechtler
Karl
K
Durbin
Richard
R
Stewart
A Francis
AF
Peters
Jan-Michael
JM
Buchholz
Frank
F
Hyman
Anthony A
AA
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004351:Drug Resistance; D005786:Gene Expression Regulation; D015723:Gene Library; D005818:Genetic Engineering; D016678:Genome; D023281:Genomics / Q000379:methods*; D008322:Mammals / Q000235:genetics* / Q000378:metabolism*; D040081:Protein Array Analysis; D011485:Protein Binding; D021381:Protein Transport; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics*
D000900:Anti-Bacterial Agents; D011506:Proteins