Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.
Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells.
Hong
Y K
YK
Harvard Institute of Human Genetics, Harvard Medical School, Beth Israel Deaconess Medical Center, 4 Blackfan Circle, Boston, MA 02115, USA.
Kim
D H
DH
Beletskii
A
A
Lee
C
C
Memili
E
E
Strauss
W M
WM
D016475:3T3 Cells; D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D005260:Female; D015870:Gene Expression / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D007372:Interferons / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics; D013234:Stem Cells; D013752:Tetracycline / Q000494:pharmacology; D015534:Trans-Activators / Q000187:drug effects; D014157:Transcription Factors / Q000235:genetics
D004251:DNA Transposable Elements; D008164:Luminescent Proteins; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D015534:Trans-Activators; D014157:Transcription Factors; C077885:XIST non-coding RNA; D049452:Green Fluorescent Proteins; D007372:Interferons; D013752:Tetracycline