Review Open Access Long range gene-regulatory sequences identiied by transgenic expression of bacterially-engineered enhancer-trap BACs in zebraish Hope M. Wolf 1,2 , Kevin O. Nyabera 1 , Katya K. De La Torre 1 , Mugtaba A. Eltayeb 1 , Oladoyin Iranloye 1 , Leighcraft A. Shakes 1 , Charles Hatcher 1 , Derek C. Norford 1 and Pradeep K. Chatterjee 1* Abstract Large pieces of DNA from the chromosomes of numerous organisms, including the human, are faithfully propagated in bacteria as large extra-chromosomal plasmids known as Bacterial Artificial Chromosomes (BACs). Because they represent tiny contiguous pieces of the chromosome, BACs are ideally suited for expression of genes in their chromosomal contexts. Genes in BACs need to be functionalized with reporter genes and other sequences that allow easy monitoring, stable maintenance and propagation of the DNA in the new host organism. BAC DNA can be altered within its bacterial host in several ways. One approach uses Tn10 mini-transposons to introduce exogenous DNA into BACs for a variety of purposes. The random insertions of Tn10 transposons carrying lox sites have helped position mammalian cell- selectable antibiotic resistance genes, enhancer-traps and inverted repeat end-sequences of the vertebrate transposon Tol2 precisely at the ends of genomic DNA inserts in BACs. Functional identification of gene- regulatory elements through reporter gene expression and BAC DNA integration into zebrafish or mouse chromosomes have been facilitated with such retrofitting. The methodology has been used extensively to dissect the regulation of the Amyloid Precursor Protein (appb) gene in zebrafish. Functional identification of long-range regulatory sequences of appb has provided important clues for regulation of the APP gene in humans. Keywords: Enhancer-trap BACs, BAC transgenesis, zebrafish transgenesis, regulation by E4BP4/NFIL3, reason for AD connection to circadian clock, alzheimer’s disease © 2014 Chatterjee et al; licensee Herbert Publications Ltd. his is an Open Access article distributed under the terms of Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0). his permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Review Using BACs for regulated expression of genes in vertebrates Proteins that bind DNA interact with different sequences with varying strengths. Histone proteins in nucleosomes do not have strong preference for binding to a specific sequence, although they exhibit subtle biases that tend to phase them appropriately with other transcription factors regulating the expression of a gene [1,2]. By contrast, proteins that recognize a specific sequence in DNA make contact not only with individual functional groups within the base pairs of the consensus sequence recognized by the protein but usually also interact weakly with sequences flanking it. Flanking sequences contribute substantially to the binding affinity of the protein to its consensus site and the interactions can sometimes extend over two turns of the helix [3,4]. Complications to phasing of both nucleosomes and proteins recognizing a specific sequence can arise from double stranded DNA that prefer to exist not as a straight helix but as curved or bent helixes in solution under physiological conditions [5]. Thus in order to mimic endogenous expression, it is important not only to have the consensus binding sites of specific transcription factors known to regulate the gene but also the proper context of these sites with regard to surrounding DNA. Regulatory proteins bound to different sites along the DNA must interact with one another to transcribe the gene, possibly in a tissue- and time-specific manner and at appropriate levels *Correspondence: pchatterjee@nccu.edu 1 Julius L. Chambers Biomedical/Biotechnology Research Institute and Department of Chemistry, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA. 2 Department of Chemistry, University of North Carolina at Chapel-Hill, Chapel-Hill, NC 27599, USA. Molecular Biology and Genetic Engineering ISSN 2053-5767 CrossMark ← Click for updates