Mice with disrupted GM2/GD2 synthase gene lack complex gangliosides but exhibit only subtle defects in their nervous system

Gangliosides, sialic acid-containing glycosphingolipids, are abundant in the vertebrate (mammalian) nervous system. Their composition is spatially and developmentally regulated, and gangliosides have been widely believed to play essential roles in establishment of the nervous system, especially in neuritogenesis and synaptogenesis. However, this has never been tested directly. Here we report the generation of mice with a disrupted f81,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase; EC 2.4.1.92) gene. The mice lacked all complex gangliosides. Nevertheless, they did not show any major histological defects in their nervous systems or in gross behavior. Just a slight reduction in the neural conduction velocity from the tibial nerve to the somatosensory cortex, but not to the lumbar spine, was detected. These findings suggest that complex gangliosides are required in neuronal functions but not in the morphogenesis and organogenesis of the brain. The higher levels of GM3 and GD3 expressed in the brains of these mutant mice may be able to compensate for the lack of complex gangliosides. Gangliosides have been considered to play important roles in the development and differentiation of the nervous system (1-3), since they are abundantly expressed in the nervous system of many vertebrates (4, 5). Gangliosides are synthesized by consecutive addition of monosaccharides to ceramide by multiple glycosyltransferases in the Golgi apparatus (6-8) and are then transferred to the outer leaflet of the plasma membrane. Among the many glycosyltransferases, 31,4 GalNActransferase (01,4 GalNAc-T; GM2/GD2 synthase; EC 2.4.1.92) plays an important role in biosynthesis of almost all complex gangliosides (Fig. 1) (9, 10, tt). Expression of this gene was markedly increased at late stages of development (12-14), suggesting roles for complex gangliosides in the neuritogenesis and synaptogenesis. To address the role of gangliosides in neuronal development and physiological events in the neural tissue, we generated mice with a disruption of the 11,4 GalNAc-T gene by homologous recombination in mouse embryonic stem (ES) cells. MATERIALS AND METHODS Gene Targeting and Generation of (81,4 GalNAc-T -/Mice. The mouse f1,4 GalNAc-T was cloned from BALB/c mouse genomic library using a 2.1-kb XbaI fragment of mouse cDNA clone pTm3-5 (15) as a probe. The targeting plasmid was constructed containing a neomycin-resistant gene inserted The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ?1734 solely to indicate this fact. into the exon 4 as shown in Fig. 2A. The targeting vector (24 nM) was linearized with NotI and was mixed with ES cell suspension (1 X 107), then electroporated at 0.25 kV, 960 tF, using a Bio-Rad Genepulser. Forty-eight hours after electroporation, G418 was added to the medium at the concentration of 150 Zg/ml. After 7-8 days, the G418-resistant clones were isolated and subjected to screening for homologous recombination by PCR. The sense primer was 5'-TCGTGCTTTACGGTATCGCCGCTCCCGATT-3' in 3' terminus of PGK neo, and the antisense primer was 5'-GGGTGTGGCGGCATACATCT-3' in the intron of the 31,4 GalNAc-T gene. The reaction was started one cycle of 95?C (2 min), 55?C (1 min), 74?C (5 min), thereafter 35 cycles of 94?C (1 min), 60?C (30 sec), 74?C (1.5 min) were used. Homologous recombinant clones gave a 1.1-kb fragment. Two chimeric males derived from ES cell lines D-120 and G-193, respectively, transmitted the 31,4 GalNAc-T mutation to progeny. Mice heterozygous for the disrupted 11,4 GalNAc-T gene were mated, and homozygous mutant progeny were identified by PCR and Southern blot analysis of DNA isolated from mouse tails. TLC and Enzyme Assay of (81,4 GaINAc-T. Glycolipids were extracted as described (16). Briefly, lipids were extracted by chloroform/methanol at ratios of 2:1, 1:1, then 1:2 sequentially. Glycolipids were isolated by a Florisil column after acetylation, then neutral and acidic fractions were separated by DEAE-Sephadex (A-50) column chromatography. The enzyme activity of 11,4 GalNAc-T was measured according to the method described (17). The membrane fractions were prepared as described by Thampoe et al. (18). The enzyme products were isolated by C18 Sep-Pak cartridge (Waters) and analyzed by TLC and fluorography as described (17). The enzyme activity of 11,3 GalNAc-T [GbOse4Cer synthase; globotetraosyl-ceramide (globoside) synthase] was measured as described (19). Neuraminidase Treatment. Neuraminidase digestion of gangliosides were performed as described (20). Morris Water-Maze and Other Behavior Tests. The mice were subjected to the Morris water-maze test for spatial learning at the age of 10 weeks as described (21). The water-maze test was conducted twice a day for 3 days. One trial consisted of starting the mice at four points 900 apart and allowing them to swim and escape onto a hidden platform in the tub. The pillar holding the platform was fixed at the center of one quadrant of the circular tub (60 cm). The top of the Abbreviations: GaINAc-T, GalNAc-transferase; ES cells, embryonic stem cells; SEP, somatosensory evoked potential; SSEP, spinal SEP; GbOse4Cer, globotetraosyl-ceramide (globoside); wt, wild type. **To whom reprint requests should be addressed. ttGanglioside nomenclature is based on that of Svennerholm (11): GM2, GalNAcJ31,4(NeuAca2, 3) Gal,Bl,4Glc-Cer; GM1, GalI31,3GaINAcJ31,4(NeuAca2, 3)GalI31,4Glc-Cer.