2006 merck/merial - School of Veterinary Medicine - Louisiana State ...

Utilizing cDNA Subtraction to Examine the Effects of MacroH2A Deletion in a Colony of Knockout FemaleMiceKatherine McKeown*, Lakshmi Changolkar, John PehrsonDepartment of Animal Biology, School of Veterinary Medicine, University of PennsylvaniaMacroH2As are histone variants with a unique structure: they are larger than the core histone H2A, having a fulllengthH2A domain on the N-terminus. macroH2As contain a C-terminal non-histone region, comprising 57% of the protein,whose function remains unclear. There are three known macroH2A subtypes: macroH2A1.1 and 1.2 are produced byalternate splicing of the same gene; macroH2A2 is coded on a separate gene. The macroH2As are highly evolutionarilyconserved, suggesting their involvement in critical functions. Studies have shown that macroH2A plays a role in maintainingX inactivation, alongside the nuclear RNA Xist. Knockout strains of mice have been produced to examine the functions ofmacroH2A, focusing on gene expression of macraH2A1 knockouts to date. Strains of knockout mice have been examined bymicroarray analysis, which identified 4 or 5 genes that are consistently changed in female livers. Microarray analysis of malemice gave similar but not identical results, suggesting sexual dimorphism in macroH2A function. Several of the genesaffected by macroH2A knockout have metabolic functions. Though microarray analysis has revealed consistent alterationslinked to macroH2A1 knockout, it has some limitations, especially regarding evaluation of rare transcripts. The objective ofthis study is to examine the effects of macroH2A1 knockout on transcription of regions not covered by the microarray.Studies have suggested that the genome contains regions that express untranslated RNAs. A notable example is the RNAXist, mentioned above. The potential expression of such RNAs, if present, are not well represented in current microarraytechniques. cDNA subtraction and PCR techniques are being used to further substantiate the results of microarray analysis, toreveal any additional genes regulated by macroH2A, and to reveal any unidentified RNAs. If successful, the results of thisexperiment will help specify the precise role that MacroH2As play in gene regulation.Methylation Status of Intracytoplasmic Sperm Injected Horse EmbryosPatsy Morris*, Y.H. Choi, and K. HinrichsDepartment of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&MUniversity, College Station, TXAn important regulator of DNA transcription and embryogenesis is methylation of DNA. The male pronuclei ofmouse, pig, rat, bovine, and human zygotes are significantly demethylated by the oocyte's cytoplasm after fertilization.However, both rabbit and sheep zygotes lack demethylation of either parental pronucleus. Thus, it is uncertain whetherspecific methylation status is necessary for further differentiation and maturation of embryos in other mammalian species,excluding the mouse. In many mammalian species, somatic nuclear transfer cloned embryos show aberrant methylationpatterns when compared to their in vivo embryos. No information has been published on the methylation status of developingembryos in the horse. The evaluation of the methylation status of intracytoplasmic sperm injected (ICSI) horse embryos atvarious stages can be used in future cloning studies. The 5-Methylcytosine (5-MeC) immunostaining protocol wasperformed on ICSI horse blastocysts, 16 hour zygotes, and embryos at day 2, day 4, and day 6, and all were shown to bemethylated. In order to establish that our primary antibody (1:50 dilution of mouse monoclonal antibody to 5-MeC) wasstaining specifically for methylated cytosine residues in the DNA as opposed to all cytosine residues, a dilution protocol wasdeveloped in which immature equine oocytes with intact cumulus cells were treated with decreasing concentrations of 5-MeCand cytosine, respectively, along with a fixed amount of primary antibody. We found that while 5-MeC binds to the primaryantibody cytosine does not; therefore, the primary antibody stains specifically for methylated cytosine residues in the DNA.The validity of our staining protocol will be tested using mouse zygotes whose methylation status is known.Modeling Neurodegenerative Disorders using the SKNAS cell line, DAT-cre, and Nestin-cre transgenic miceKara Myslivec*, Kate Jackson, Holli Charbonneau, Dr. James Resau, Dr. Bart WilliamsSupported in part by the Merck-Merial Veterinary Scholars Program at Michigan State University, and the VanAndel Research InstituteParkinson’s disease (PD) is a common disabling neurodegenerative disorder primarily affecting individuals betweenthe ages of 50 and 60, although young adults can also be affected. The death of dopaminergic neurons in the substantia nigrapars compacta (SNpc) is the major cause of the symptoms. Dopamine is produced in the SNpc and transported to the striatumwhere it regulates motor function. Certain genetic and environmental factors may also induce ‘parkinsonism’ via mitochondrialdysfunction and oxidative stress. About 95% of cases are sporadic, while 5% of cases are attributed to a genetic predispositionto PD. The genetic predispositions are associated with specific mutations in primarily the mitochondria, ubiquitin and metabolicsystems. These cases show familial clustering and typically have an earlier onset of symptoms. We will use animals of knowngenotype, such as DAT-cre and Nestin-cre knock-out mice to affect pathways in specific areas of the dopaminergic neurons andneuronal precursor cells, respectively. In addition, we will also utilize SKNAS cells, a neuroblastoma cell line for an in vitromodel. The mice will be treated with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and the cell lines will be treated146