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Title: Identification of essential roles for N-linked carbohydrates in mouse development
Authors: Ioffe, Ella
Keywords: Molecular biology.
Cellular biology.
Issue Date: 1995
Publisher: ProQuest Dissertations & Theses
Citation: Source: Dissertation Abstracts International, Volume: 56-10, Section: B, page: 5320.
Abstract: It has been established that eukaryotic cells absolutely require N-linked carbohydrates for survival. However, the truncated structure Man{dollar}\sb5{dollar}GlcNAc{dollar}\sb2{dollar}Asn, in place of mature N-linked carbohydrates, allows glycoprotein synthesis and somatic cell growth to proceed normally. The cloning of the Mgat1 gene encoding N-acetylglucosaminyltransferase I (GlcNAc-TI) that initiates the synthesis of mature, N-linked carbohydrates from Man{dollar}\sb5{dollar}GlcNAc{dollar}\sb2{dollar}Asn, provided the possibility of determining whether these N-linked carbohydrates would be required in a complex biological situation, such as the developing mouse.;The approach was to disrupt the Mgat1 gene encoding N-acetylglucosaminyltransferase I in ES cells through homologous recombination and to transmit this mutation to the mouse germline. Mgat1 +/{dollar}-{dollar} heterozygotes appeared completely normal in growth, development and fertility. However, matings between heterozygotes did not give rise to any homozygous {dollar}-/-{dollar} progeny. Staining of preimplantation and early postimplantation mutant embryos with the lectin L-PHA, that specifically binds complex N-linked carbohydrates, revealed that complex N-glycans are present in mutant blastocysts at embryonic day 3.5, but they are not present at day 6.5. PCR/RT PCR analyses of mutant blastocysts proved that maternal Mgat1 gene product is present in these embryos. Biochemical and morphological analyses of embryos from 8.5 to 13.5 days of development demonstrated that Mgat1 {dollar}-/-{dollar} embryos are developmentally retarded and that they die between 9.5 and 10.5 days of gestation.;Two independently isolated Mgat1 {dollar}-/-{dollar} ES cell lines, that cannot produce complex and hybrid N-linked carbohydrates, were shown to grow and differentiate normally in culture, but had an increased potential to form malignant neural tissues. These ES cell lines formed good chimeras, but they specifically did not colonize bronchial epithelium of 16.5 day mouse lungs. This phenotype was corrected by transfection of Mgat1 {dollar}-/-{dollar} mutant ES cells with an Mgat1 coding region genomic DNA fragment demonstrating that the lung phenotype was specifically due to the lack of GlcNAc-TI activity.
Appears in Collections:Albert Einstein College of Medicine: Doctoral Dissertations

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