A Practical Approach, Second Edition=Ronald D. Ho.pdf

624 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITION1. The DNA deposited on the solid support can be easily prepared by investigators using standardPCR amplification.2. For producers of their own arrays, sets of cloned genes can be purchased from biotechnologycompanies, obtained from the National Institutes of Health (NIH), or prepared in-house. Thus,researchers have a great deal of control over the content of the arrays they make and use.3. The cost of preparing cDNA arrays in-house is substantially lower than the cost of commerciallyavailable arrays. Once the basic equipment for array fabrication has been obtained and the targetDNA prepared, hundreds of microarray slides can be produced in a relatively short time.a. Array FabricationThe choice of which genes or expressed sequence tags (ESTs) to use for cDNA array fabricationdepends on their availability; however, all deposited target DNA is prepared by standard PCRamplification of cDNA cloned into bacterial plasmids. Individually cloned genes and ESTs comein sets of several hundred to several thousand. They are either commercially available, obtainedfree of charge (e.g., from the NIH), or they may even be developed within the institution. SeeFigure 15.1 for an outline of cDNA microarray fabrication.Comprehensive collections of genes and ESTs are particularly useful for applications such asgene discovery, when the goal is to associate specific genes with a specific toxicological process.Several cDNA clone sets are commercially available. 10 These include: the BMAP clone set (Res-Gen, Carlsbad, CA), which was derived from adult C57BL6J mouse brain, spinal cord, and retinaltissues; arrayTAG (Lion Biosciences, San Diego, CA), which is a family of cDNA clones (mouse,rat, and dog) that were specifically designed for microarray applications by use of several uniquemethodologies; the full-length mouse cDNA clones from the RIKEN Genomic Sciences Center(Yokohama, Japan); and more.Two of the most useful clone sets for developmental biologists and developmental toxicologistsalike, the mouse 15K and 7.4K sets, were assembled at the National Institute of Aging. The 15Kclone set was constructed to specifically represent developmentally important mammalian genes. 11,12The approximately15,000 genes in this set were derived from pre- and peri-implantation embryosand from placenta. Between a third to a half of the 15,000 set contains known and previouslycharacterized genes, whereas the reminder is composed of ESTs of currently unknown function. 11The second clone set, released in 2002, is the 7400 clone set that contains approximately 7400genes with no redundancy within the set or with the original 15,000 set. The 7400 clone set isespecially valuable for developmental biologists and toxicologists as it includes genes expressedin a variety of stem cells, early embryos, and newborn organs, such as the heart and brain.Preparing in-house clone sets requires considerable molecular biology expertise and resources;however, it enables researchers to construct microarray slides with a tissue-specific set of genes orwith genes that are expressed during specific developmental processes, such as palatal shelf fusion.Using the in-house approach, investigators studying eye development created a library of approximately15,000 ESTs that were obtained from mouse retina at embryonic day 14.5. 13 By use ofmicroarray slides that were prepared from this clone set, a potential regulatory association betweenthe transcription factor Brn-3b and GAP-43, a protein associated with axonal growth, has beenidentified.Similarly, at the National Center for Toxicogenomics (NCT), which is part of the NationalInstitute of Environmental Health Sciences (NIEHS), a toxicological human cDNA chip called theToxChip has been developed. 14,15 The 2000 genes arrayed on the early version of the ToxChip(version 1.0) have been previously implicated in mechanisms of toxicity and cellular processes inresponse to different types of toxic insults. The latter include genes involved in apoptosis, DNArepair, and drug metabolism, transcription factors, tumor suppressor genes, and more. A similarapproach was used to evaluate the effects of the environmental toxicant β-naphthoflavone on miceby use of a toxicological gene array and Northern blotting. 16 The authors compared the two methods© 2006 by Taylor & Francis Group, LLC