The cis-regulatory element Gsl5 is indispensable for proximal straight tubule cell-specific transcription of core 2 beta-1,6-N-acetylglucosaminyltransferase in the mouse kidney.

Gsl5 regulates the expression of a glycolipid and glycoproteins that contain the Le(X) epitope in the mouse kidney through tissue-specific transcriptional regulation of the core 2 beta-1,6-N-acetylglucosaminyltransferase (core 2 GnT) gene. The core 2 GnT gene has six exons and produces three alternatively spliced transcripts. Gsl5 regulates only the expression of the kidney-type mRNA, which is transcribed from the most 5'-upstream exon. By introducing a 159-kb bacterial artificial chromosome (BAC) clone that carries the mouse core 2 GnT gene and its 5'-upstream region into DBA/2 mice that carry a defective Gsl5 allele, we were able to rescue the deficient phenotype. The BAC clone was subsequently engineered to replace the core 2 GnT gene with the sequence of enhanced green fluorescent protein (EGFP) as a reporter by an inducible homologous recombination system in Escherichia coli. The transgenic mice derived from the modified BAC clone expressed EGFP in the kidney, which suggests that the candidate Gsl5 is in the 5'-upstream region of the core 2 GnT gene. Sequence analysis of the 5'-upstream regions of the BAC clone and DBA/2 genomic DNA revealed a candidate sequence for Gsl5 at about 5.5 kb upstream of exon 1. This sequence consisted of eight repeats of two GT-rich units in the wild-type mice, whereas it consisted of only one pair of GT-rich units with a minor modification in the DBA/2 mice. Transgenic mice produced with the EGFP reporter gene construct that included this candidate sequence expressed EGFP exclusively in the proximal straight tubular cells of the kidney. These results indicated that this unique repeat is indeed the Gsl5, and it is a cis-regulatory element responsible for proximal straight tubule cell-specific transcriptional regulation.

Expansion of natural killer cells in mice transgenic for IgM antibody to ganglioside GD2: demonstration of prolonged survival after challenge with syngeneic tumor cells.

IgM antibodies to gangliosides, sialic acid-containing glycosphingolipids, have been shown to mediate anti-tumor effects in cancer patients with melanoma and neuroblastoma and to correlate with survival. Mechanisms by which the antibodies induce tumor suppression, however, have not been systematically studied. To investigate this point, we produced and characterized C57BL/6 mice transgenic for IgM antibody to ganglioside GD2. The transgenic (TG) mice showed high IgM, but not IgG antibody titers against GD2 in their sera. No significant clinical symptoms were observed. When EL4 cells, syngeneic T lymphoma that express ganglioside GD2, were injected into TG mice, prolonged survival was observed. Complement-dependent cytotoxicity (CDC) of EL4 cells was mediated with TG mice sera. Neither antibody-dependent cellular cytotoxicity with their sera nor cytotoxic T lymphocyte activity to EL4 cells was shown in TG mice. Spleen lymphocytes from TG mice had increased numbers of natural killer (NK) cells, but not T cells, B cells, or macrophages compared with wild-type mice. Depletion of NK cells with anti-asialo GM1 rabbit serum reduced or abrogated the observed anti-tumor effects, suggesting that NK cells play a major role in tumor eradication or suppression. NK cell activity in TG mice was much higher than wild-type mice. Moreover, TG mice showed prolonged survival after injection with syngeneic B16 melanoma cells, which express GM3, but not GD2 or GD3. Taking these results together, our studies demonstrate that the TG mice have significant anti-tumor characteristics, probably due to CDC and NK cell expansion and activation with anti-ganglioside GD2 antibody.