Abstract
BAC transgenesis in mice has proved to be useful in exploring the regulatory mechanisms and functions of the Hox complexes. The large constructs used may include most of the relevant components of the cis-regulatory landscape. Manipulations can be accomplished without compromising the integrity of the endogenous complex which reduces the likelihood of producing confounding phenotypic abnormalities. The development of recombineering tools has been critical in providing the means necessary to make many types of precise and varied manipulations of these large constructs. Here, we will discuss the methodologies necessary to manipulate Hox complex BACs, generation of transgenic animals bearing these constructs and the utilization of these resources to address fundamental aspects of Hox biology.
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References
Sharpe J, Nonchev S, Gould A et al (1998) Selectivity, sharing and competitive interactions in the regulation of Hoxb genes. EMBO J 17(6):1788–1798
Barrow JR, Capecchi MR (1996) Targeted disruption of the Hoxb-2 locus in mice interferes with expression of Hoxb-1 and Hoxb-4. Development 122(12):3817–3828
Parrish M, Unruh J, Krumlauf R (2011) BAC modification through serial or simultaneous use of CRE/Lox technology. J Biomed Biotechnol 2011:924068
Ellis HM, Yu D, DiTizio T et al (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci U S A 98(12):6742–6746
Copeland NG, Jenkins NA, Court DL (2001) Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet 2(10):769–779
Lee EC, Yu D, Martinez de Velasco J et al (2001) A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73(1):56–65
Warming S, Costantino N, Court DL et al (2005) Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 33(4):e36
Pikaart MJ, Recillas-Targa F, Felsenfeld G (1998) Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators. Genes Dev 12(18):2852–2862
Ahn Y, Mullan HE, Krumlauf R (2014) Long-range regulation by shared retinoic acid response elements modulate dynamic expression of posterior Hoxb genes in CNS development. Developmental Biology 388(1): 134–144.
Muyrers JP, Zhang Y, Benes V et al (2000) Point mutation of bacterial artificial chromosomes by ET recombination. EMBO Rep 1(3):239–243
Blomfield IC, Vaughn V, Rest RF et al (1991) Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol Microbiol 5(6):1447–1457
Zhang Y, Buchholz F, Muyrers JP et al (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20(2):123–128
Nolte C, Jinks T, Wang X et al (2013) Shadow enhancers flanking the HoxB cluster direct dynamic Hox expression in early heart and endoderm development. Dev Biol 383(1):158–173
Acknowledgements
We thank members of the Krumlauf lab for valuable discussions, Heidi Monnin, Jenny Renyolds, and Andrea Moran and other members of the Stowers Laboratory Animal Services Facility for their care of our mice and technical assistance. All experiments involving mice were approved by the Institutional Animal Care and Use Committee of the Stowers Institute for Medical Research (Protocol 2010-0062/2013-0114). This research was supported by funds from the Stowers Institute and in addition Mark Parrish was funded in part by a Ruth L. Kirschstein NRSA Postdoctoral Fellowship (1F32GM075611-01/5F32GM075611-02).
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Parrish, M., Ahn, Y., Nolte, C., De Kumar, B., Krumlauf, R. (2014). Hox Complex Analysis Through BAC Recombineering. In: Graba, Y., Rezsohazy, R. (eds) Hox Genes. Methods in Molecular Biology, vol 1196. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1242-1_5
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DOI: https://doi.org/10.1007/978-1-4939-1242-1_5
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