Nuclear and cytoplasmic protein glycosylation is a widespread and reversible posttranslational modification in eukaryotic cells. Intracellular glycosylation by the addition of N-acetylglucosamine (GlcNAc) to serine and threonine is catalyzed by the O-GlcNAc transferase (OGT). This ''O-GlcNAcylation'' of intracellular proteins can occur on phosphorylation sites, and has been implicated in controlling gene transcription, neurofilament assembly, and the emergence of diabetes and neurologic disease. To study OGT function in vivo, we have used gene-targeting approaches in male embryonic stem cells. We find that OGT mutagenesis requires a strategy that retains an intact OGT gene as accomplished by using Cre-loxP recombination, because a deletion in the OGT gene results in loss of embryonic stem cell viability. A single copy of the OGT gene is present in the male genome and resides on the X chromosome near the centromere in region D in the mouse spanning markers DxMit41 and DxMit95, and in humans at Xq13, a region associated with neurologic disease. OGT RNA expression in mice is comparably high among most cell types, with lower levels in the pancreas. Segregation of OGT alleles in the mouse germ line with ZP3-Cre recombination in oocytes reveals that intact OGT alleles are required for completion of embryogenesis. These studies illustrate the necessity of conditional gene-targeting approaches in the mutagenesis and study of essential sex-linked genes, and indicate that OGT participation in intracellular glycosylation is essential for embryonic stem cell viability and for mouse ontogeny.I ntracellular protein glycosylation is ubiquitous in eukaryotic cells yet is less studied than other types of posttranslational modifications such as phosphorylation. This is, in part, because of the relatively recent discovery that serine and threonine residues of many cytoplasmic and nuclear proteins are modified by the addition of an O-linked N-acetylglucosamine (O-GlcNAc) (1, 2). Moreover, this modification originally was difficult to detect before the development of new approaches. O-GlcNAc formation, also termed O-GlcNAcylation, has been found on nuclear pore proteins, RNA polymerase II, and many transcription factors, including Sp1, serum response factor, and the estrogen receptors. In addition, cytoskeletal proteins such as Tau, vinculin, talin, ankyrin, neurofilaments, cytokeratins, and clathrin assembly protein AP-3; and viral or oncogene proteins, such as the cytomegalovirus UL32 protein, myc, fos, jun, simian virus 40 T-antigen, and p53 are also .UDP-N-acetyl-D-glucosamine:protein ␤-N-acetyl-D-glucosaminyltransferase (OGT) (EC 2.4.1.94) catalyzes the addition of O-GlcNAc to the polypeptide chain. All higher eukaryotic cell types studied contain OGT activity. Purified rat liver OGT is a heterotrimer with two catalytic subunits of 110 kDa and a 78-kDa subunit of unknown function (22). The 78-kDa form is structurally and immunologically related to the 110-kDa protein and may result from an internal translation start site or fro...

We have developed a method of specifically modifying the mammalian genome in vivo. This procedure comprises heritable tissue-specific and site-specific DNA recombination as a function of recombinase expression in transgenic mice. Transgenes encoding the bacteriophage P1 Cre recombinase and the loxP-flanked fi-galactosidase gene were used to generate transgenic mice. Genomic DNA from doubly transgenic mice exhibited tissue-specific DNA recombination as a result of Cre expression. Further characterization revealed that this process was highly efficient at distinct chromosomal integration sites. These studies also imply that Cre-mediated recombination provides a heritable marker for mitoses following the loss of Cre expression. This transgene-recombination system permits unique approaches to in vivo studies of gene function within experimentally defmed spatial and temporal boundaries.Bacteriophage P1 encodes the 38-kDa Cre recombinase that catalyzes site-specific DNA recombination between 34-basepair (bp) repeats termed loxP (1). Cre is a member of the integrase family of recombinases. These enzymes recognize specific nucleotide sequences and function through a transient DNA-protein covalent linkage (reviewed in refs. 2 and 3). Cre activity appears mechanistically identical to that of yeast FLP recombinase and can function in vitro in the absence of high-energy cofactors, topoisomerase activity, and DNA replication (4, 5). In Cre-mediated recombination, resultant DNA structures are dependent upon the orientation of loxP sites. Direct repeats of loxP dictate an excision of intervening sequences whereas inverted repeats specify inversion (4). Cre and FLP have been shown to mediate site-specific DNA recombination in tissue-cultured eukaryotic cells, Drosophila, and transgenic plants (6)(7)(8)(9)(10)(11)(12).With the aim of applying Cre recombinase function to molecular studies of normal and abnormal mammalian physiology, we sought to generate a transgenic mouse system that would establish whether Cre could effectively mediate chromosomal DNA recombination. As a foundation for future applications, we devised a nondeleterious transgene strategy that would provide an assessment of the efficiency, position dependence, and heritability of Cre-mediated chromosomal DNA recombination in mammals.

To test whether kinesin-II is important for transport in the mammalian photoreceptor cilium, and to identify its potential cargoes, we used Cre-loxP mutagenesis to remove the kinesin-II subunit, KIF3A, specifically from photoreceptors. Complete loss of KIF3A caused large accumulations of opsin, arrestin, and membranes within the photoreceptor inner segment, while the localization of alpha-transducin was unaffected. Other membrane, organelle, and transport markers, as well as opsin processing appeared normal. Loss of KIF3A ultimately caused apoptotic photoreceptor cell death similar to a known opsin transport mutant. The data suggest that kinesin-II is required to transport opsin and arrestin from the inner to the outer segment and that blocks in this transport pathway lead to photoreceptor cell death as found in retinitis pigmentosa.