title: Making BAC transgene constructs with lambda-red recombineering system for transgenic animals or cell lines.
abstract: The genomic DNA libraries based on Bacteria Artificial Chromosomes (BAC) are the foundation of whole genomic mapping, sequencing, and annotation for many species like mice and humans. With their large insert size, BACs harbor the gene-of-interest and nearby transcriptional regulatory elements necessary to direct the expression of the gene-of-interest in a temporal and cell-type specific manner. When replacing a gene-of-interest with a transgene in vivo, the transgene can be expressed with the same patterns and machinery as that of the endogenous gene. This chapter describes in detail a method of using lambda-red recombineering to make BAC transgene constructs with the integration of a transgene into a designated location within a BAC. As the final BAC construct will be used for transfection in cell lines or making transgenic animals, specific considerations with BAC transgenes such as genotyping, BAC coverage and integrity as well as quality of BAC DNA will be addressed. Not only does this approach provide a practical and effective way to modify large DNA constructs, the same recombineering principles can apply to smaller high copy plasmids as well as to chromosome engineering.
authors.affiliation: The Neuroscience Center, University of North Carolina at Chapel Hill, 115 Mason Farm Road, Chapel Hill, NC, 27599, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Holmes
authors.forename: Suzanne
authors.initials: S
authors.lastname: Lyman
authors.forename: Jen-Kang
authors.initials: JK
authors.lastname: Hsu
authors.forename: JrGang
authors.initials: J
authors.lastname: Cheng
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D018274:Electroporation; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D005697:Galactosides / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D015698:Genomic Library; D059765:Homologous Recombination; D007211:Indoles / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009876:Operon; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D019076:Transgenes
chemical_list: D004269:DNA, Bacterial; D005697:Galactosides; D007211:Indoles; D007544:Isopropyl Thiogalactoside; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside