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Echinobase
ECB-ART-49929
Methods Cell Biol 2019 Jan 01;151:197-218. doi: 10.1016/bs.mcb.2019.01.004.
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Techniques for analyzing gene expression using BAC-based reporter constructs.



Abstract
To characterize the complex regulatory control of gene expression using fluorescent protein reporters, it is often necessary to analyze large genomic regions. Bacteria artificial chromosome (BAC) vectors, which are able to support DNA fragments of up to 300kb, provide stable platforms for experimental manipulation. Using phage-based systems of homologous recombination, BACs can be efficiently engineered for a variety of aims. These include expressing fluorescent proteins to delineate gene expression boundaries using high-resolution, in vivo microscopy, tracing cell lineages using stable fluorescent proteins, perturbing endogenous protein function by expressing dominant negative forms, interfering with development by mis-expressing transcription factors, and identifying regulatory regions through deletion analysis. Here, we present a series of protocols for identifying BAC clones that contain genes of interest, modifying BACs for use as reporter constructs, and preparing BAC DNA for microinjection into fertilized eggs. Although the protocols here are tailored for use in echinoderm embryonic and larval stages, these methods are easily adaptable for use in other transgenic systems. As fluorescent protein technology continues to expand, so do the potential applications for recombinant BACs.

PubMed ID: 30948008
PMC ID: PMC7215881
Article link: Methods Cell Biol
Grant support: [+]


References [+] :
Barsi, General approach for in vivo recovery of cell type-specific effector gene sets. 2014, Pubmed, Echinobase