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

CELLULAR, BIOCHEMICAL, AND MOLECULAR TECHNIQUES IN DEVELOPMENTAL TOXICOLOGY 613rate. Examination of the transcription rate by this method suggested that the effects of the chemicalexposure were at a posttranscriptional level. 57 The embryonal carcinoma cells also express PDGFα-receptor, and the combination of retinoic acid and dibutyryl cyclic AMP stimulated expressionof mRNA for that receptor. 58 The PDGF growth factors are expressed in embryos, and a lethalmutation in the patch locus correlates with failure to express the PDGF α-receptor. Exploring theregulation of this receptor with Northern blots and nuclear-run-on analyses demonstrated stronginduction by the RA-cAMP treatment. The study also showed that transcriptional rates increasedproportionally with mRNA levels, indicating transcriptional regulation was involved in the response.IV. CHARACTERIZING THE ROLE OF SPECIFIC GENESAND PATHWAYS OF RESPONSEMolecular techniques can be helpful in identifying potential biomolecules involved in response toa teratogen. This approach is conceptually the inverse of that discussed previously. In this alternativecase, the approach is to screen the genome of the embryo. Differences between treated and controlembryonic gene expression are determined, and a survey across the developmental period of interestmay be involved. The scope of these studies must of necessity be wider, and thus there is thepotential to identify contributors to a response that would otherwise go unrecognized. One approachinvolves screening of genetic libraries derived from embryos or specific organs in various stagesof development. Restriction fragment length polymorphism (RFLP) analysis has also been valuablein studies of human diseases and birth defects and has identified links between specific genes andmalformations. 59,60 Mutational analysis and targeted gene disruption allow the phenocopy approachto be used, in which a defect induced by a teratogenic agent can be compared to similar defectscaused by mutation or targeted gene disruption. Although potentially valuable for developmentaltoxicology studies, the RFLP and phenocopy approaches are not presented here. The followingdiscussion focuses on two methods, subtractive screening and in situ transcription RT-PCR reverseSouthern blot, which have the potential to detect differential gene expression across developmentand between experimental treatments.A. Transgenic AnimalsTransgenic mice are currently being generated to examine the regulation, function, and expressionpatterns of a wide range of genes that are implicated in controlling embryonic development. Thesetransgenic animals can be “reporter mice” that allow the expression of a particular gene to bemonitored during various stages of development. Reporter mice typically use the regulatorysequences from a gene of interest to drive the expression of a reporter gene, such as lacZ, to whichthe regulatory sequences are fused. This reporter transgene is activated in the regions that haveendogenous gene expression and is detected by the appearance of dark blue staining caused byβ-galactosidase activity.Other transgenic mice are generated to “knock out” expression of the gene of interest, generallythrough targeted mutation that results in a homozygous inactivation. These loss-of-function mutantsare produced by disruption of the gene by homologous recombination in embryonic stem (ES)cells. The mutant ES cells are then introduced into a mouse blastocyst to generate chimeric embryosthat are then placed in the hormonally primed uterus of a host, pseudopregnant female. The neonateswill be chimeric, and coat color is usually employed (host blastocyst being from the albino andES cells being from the pigmented strain) as a means of readily identifying the transgenic animals(ES cell homologous recombination was reviewed by Capecchi 61 ). The chimeric mice are bred togenerate mice homozygous for the disrupted gene, and the phenotype of the resulting embryos canbe informative concerning the role of that gene in development.© 2006 by Taylor & Francis Group, LLC