Analysis of imprinting in mice with uniparental duplication of proximal chromosomes 7 and 15 by use of a custom oligonucleotide microarray.
We have developed an imprinting assay combining the use of mice carrying maternal or paternal duplication of chromosomal regions of interest with custom oligonucleotide microarrays. As a model system, we analyzed RNA from CNS tissue of neonatal mice carrying the reciprocal translocation T(7;15)9H and uniparental duplication of proximal Chr 7 and 15. The duplicated region includes the locus on proximal Chr 7 corresponding to the human Prader-Willi/Angelman Syndrome. The microarray contained 322 oligonucleotides, including probes to detect major genes involved in neural excitability and synaptic transmission, as well as known imprinted genes mapping to proximal Chr 7: Ndn, Snrpn, Mkrn3, Magel2, Peg3, and Ube3a. Imprinting of these genes in neonatal cortex and cerebellum was first confirmed by quantitative RT-PCR. Their inclusion on the microarray thus provided positive controls for evaluating the effect of background on the sensitivity of the assay, and for establishing the minimum level of expression required to detect imprinting. Our analysis extended previous work by revealing bi-allelic expression in CNS tissue of those queried genes mapping to proximal Chr 7 or 15, including the Gabrb3 gene, for which there have been conflicting reports. Microarray analysis also revealed no effect of the maternal or paternal disomy on expression levels of the unlinked genes detected, including those potentially implicated in the Prader-Willi or Angelman Syndrome. In addition, quantitative RT-PCR revealed a gene dosage effect in both cerebellum and cortex for all of the known imprinted genes assayed, except for Ube3a in cerebellum.
Buettner
Victoria L
VL
Division of Biology, Beckman Research Institute, City of Hope, CA 91010, USA.
Longmate
Jeffrey A
JA
Barish
Michael E
ME
Mann
Jeffrey R
JR
Singer-Sam
Judith
J
D000818:Animals; D002490:Central Nervous System / Q000378:metabolism; D017931:DNA Primers; D018628:Gene Dosage; D015870:Gene Expression; D018392:Genomic Imprinting / Q000235:genetics*; D051379:Mice / Q000235:genetics*; D008817:Mice, Mutant Strains; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D024182:Uniparental Disomy / Q000235:genetics*
D017931:DNA Primers; D012313:RNA