Bacterial artificial chromosomes as analytical basis for gene transcriptional machineries.
Bacterial Artificial Chromosomes (BACs) had been minimal components of various genome-sequencing projects, constituting perfect analytical basis for functional genomics. Here we describe an enhancer screening strategy in which BAC clones that cover any genomic segments of interest are modified to harbor a reporter cassette by transposon tagging, then processed to carry selected combinations of gene regulatory modules by homologous recombination mediated systematic deletions. Such engineered BAC-reporter constructs in bacterial cells are ready for efficient transgenesis in mice to evaluate activities of gene regulatory modules intact or absent in the constructs. By utilizing the strategy, we could speedily identify a critical genomic fragment for spatio-temporally regulated expression of a mouse cadherin gene whose structure is extraordinarily huge and intricate. This BAC-based methodology would hence provide a novel screening platform for gene transcriptional machineries that dynamically fluctuate during development, pathogenesis and/or evolution.
Department of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Ogawahigashi 4-1-1, Kodaira, Tokyo, 187-8502, Japan.
Junko
J
Asami
Yukiko U
YU
Inoue
Youhei W
YW
Terakawa
Saki F
SF
Egusa
Takayoshi
T
Inoue
D000818:Animals; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics; D020869:Gene Expression Profiling; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors; D016678:Genome; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic
D015820:Cadherins; D004251:DNA Transposable Elements