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

616 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITIONis generally not as likely to detect transcripts from rarely expressed genes or to discern differencesbetween low-level expression mRNAs from a contrasted pair of samples. Subtractive hybridizationcross-hybridizes two mRNA or DNA samples that have been isolated from different experimentalsamples and then isolates the nonhybridized sequences. All nucleic acid sequences occurring inboth samples hybridize and are removed, leaving behind the unmatched mRNA or DNA. Thisisolated material represents the genes expressed in one sample and not the other. Using a higherconcentration of one sample than the other results in detection of either increased or decreasedexpression depending on which sample (control or treated, or different developmental stages) waspresent in excess.Branch et al. 80 adapted this approach to examine the effects of restraint of pregnant mice ongene expression in the embryos. Maternal restraint significantly increases the incidence of lumbarribs and encephalocele in mice. GD 8 embryonic mRNA was isolated from the treated group andreverse transcribed to give single stranded cDNA with c-tailed 3′ ends. Control mRNA wasbiotinylated and then cross-hybridized to the cDNA. Streptavidin binding removes all biotinylatedmRNA (hybridized and single stranded), subtracting or leaving behind the unmatched cDNA ofthe treated embryos. After second strand synthesis, this cDNA can be PCR amplified and clonedinto vectors for transformation of E. coli. The bacterial colonies with vector containing the cDNAinsert are isolated and sequenced, with the intent to identify the gene by comparing the sequenceto those in a gene data bank. In the stress-restraint study, seven cDNAs were isolated. One wasidentified as encoding a protein important in cell cycle regulation, and the others are under furtherstudy.Subtractive hybridization was also used to isolate mRNAs associated with programmed celldeath induced by exposure of thymoctyes to glucocorticoids. 81 One of the isolated genes wassequenced and found to contain a zinc-finger domain, suggesting involvement in DNA regulation,while a second gene appeared to encode a membrane-bound protein.When the isolated sequence is compared with known gene sequences, the isolation of a sequencethat is differentially expressed may lead to identification of the gene involved. The cDNA clonemay also be used to construct fusion proteins that can be synthesized in E. coli, purified, and usedto prepare antibodies. These antibodies may then be useful in isolating the protein that is encodedby the gene. Alternatively, the clone sequence may be used to synthesize a small peptide againstwhich to raise antibody.E. Reverse Southern Blots and RT-PCR In Situ TranscriptionIn situ transcription of mRNAs on a sectioned embryo coupled with RT-PCR and reverse Southernblotting has recently been proposed as a screen of differential gene activity. 52,82–84 With this method,specific regions of a tissue may be examined and compared with like regions from other developmentalstages, or controls can be compared with treated embryos. The in situ transcription usesoligo primers incubated directly on the sectioned tissue (or selected portion of the section followingremoval of unwanted regions) to synthesize cDNA directly from mRNA. Following separation ofthe mRNA-cDNA hybrids and second strand synthesis to generate double stranded cDNA, thismaterial can be amplified by PCR for use as a probe or it can be cloned into a vector. The riboprobesprepared from this cDNA can be used to screen Southern dot or slot blots, in which cDNA sequencesof genes suspected to be involved in the mechanism of toxicity or the developmental pathway forthat tissue are bound to the membrane. Hybridization intensity reflects the relative abundance ofthe mRNAs of interest. Gene expression in the Splotch mutant mouse, a model for neural tubedefects, was examined for a range of growth factors, transcription factors, Hox genes, and extracellularproteins. 82 This approach has detected effects on candidate genes after exposure to valproicacid, retinoic acid, or hyperthermia in SWV mice. 83 Similar studies examined changes in geneexpression after exposure of SWV mice to phenytoin. 84© 2006 by Taylor & Francis Group, LLC