Epitope tagging of proteins at the native chromosomal loci of genes in mice and in cultured vertebrate cells.
Adding epitope tags to proteins is an important method for biochemical analyses and is generally accomplished in metazoan cells using ectopically expressed, tagged trans-genes. In Saccharomyces cerevisiae, the addition of epitope tags to proteins is easily achieved at the genomic locus of a gene of interest due to the high efficiency of homologous recombination in that organism. Most metazoan cells do not exhibit this high homologous recombination efficiency, and therefore trans-genes with in-frame epitope tags are used. Although epitope tagged trans-genes have proven useful, replacing the native promoter with a heterologous promoter introduces numerous artifactual possibilities. These include overexpression, which can lead to promiscuous interactions, and the loss of native transcriptional control, which in live animals often leads to developmental defects and embryonic lethality. We describe an efficient method that overcomes the problems encountered using epitope tagged trans-genes by introducing the epitope tag into the native chromosomal gene locus in vertebrate cells, embryonic stem cells and live mice. These tagged proteins are physically associated with the expected relevant particles, and highly sensitive as shown by co-purification of homologues of the yeast pre-mRNA splicing factors Prp38p and Prp39p, not previously shown to be associated with metazoan snRNPs. These techniques will enhance the validity of conclusions made regarding epitope-tagged proteins and improve our understanding of proteomic dynamics in cultured vertebrate cells and live animals.
Chen
Yen-I G
YI
Graduate Program in Microbiology, University of Texas at Austin, 1 University Station #A4800, 2500 Speedway, Austin, TX 78712, USA.
Maika
Shanna D
SD
Stevens
Scott W
SW
D000595:Amino Acid Sequence; D000818:Animals; D001755:Blastocyst / Q000378:metabolism; D002478:Cells, Cultured; D002645:Chickens; D002875:Chromosomes / Q000235:genetics / Q000378:metabolism*; D033481:Chromosomes, Mammalian / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018388:Codon, Terminator; D018604:Epitope Mapping; D000939:Epitopes; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D000072260:RNA Splicing Factors; D011995:Recombination, Genetic; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D018388:Codon, Terminator; D000939:Epitopes; C076234:PRP38 protein, S cerevisiae; C087357:Prp39 protein, S cerevisiae; D000072260:RNA Splicing Factors; D017412:Ribonucleoprotein, U1 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C411898:Sart1 protein, mouse; D015534:Trans-Activators