Background Donor organ rejection continues to be a significant problem for patients receiving transplants. We therefore tested whether transferring a donor＇s major histocompatibility complex （MHC） gene to the recip...Background Donor organ rejection continues to be a significant problem for patients receiving transplants. We therefore tested whether transferring a donor＇s major histocompatibility complex （MHC） gene to the recipient would mitigate the rejection of transplanted hearts in mice. Methods H-2K^kgene from donor mice was amplified using nested polymerase chain reaction （PCR） and ligated into a mammalian expression vector, which was then transfected into thymus ground mass cells collected from the recipients. Clones stably expressing the transgene were then injected into the recipients＇ thymus visualized using ultrasound. Control mice were administered cells previously transfected with empty vector. Following heart transplantation, cardiac activity was monitored electrocardiographically. Recipient thymus cells were tested for MHC antigenicity using flow cytometry and spleen cells were subjected to mixed lymphocyte culture tests. Finally, the transplanted hearts were sectioned, stained and examined under light microscopy. Results Southern analysis following nested PCR revealed clear expression of H-2K^ gene. Following transplantation, electrocardiosignals were detectable highly significantly longer in recipients administered thymal cells expressing donor H-2K^ than in those receiving control cells. Flow cytometric analysis using an anti-H-2K^ antibody confirmed its expression in H-2K^ treated recipients but not in control mice. Mixed lymphocyte cultures containing H-2K^ treated cells showed significantly less proliferation than those containing control cells. Hearts from control mice showed substantially greater lymphocyte infiltration than those from H-2K^ treated mice and large areas of necrosis. Conclusion Rejection of transplanted hearts can be mitigated substantially by introducing the donor＇s MHC into the recipient.展开更多
Even though the nitrate assimilation operon has been extensively studied in Phormidium laminosum, some aspects still remain unclear. The genetic manipulation of this cyanobacterium is problematic that hinders the eluc...Even though the nitrate assimilation operon has been extensively studied in Phormidium laminosum, some aspects still remain unclear. The genetic manipulation of this cyanobacterium is problematic that hinders the elucidation of further aspects of nitrogen metabolism. To circumvent this, Thermosynechococcus elongatus BP-1 was selected as a surrogate host and its nirA gene was substituted by the homologous gene of P. laminosum. This process, based on Long Flanking Homology Polymerase Chain Reaction and the natural competence of T. elongatus BP-1, required an intermediate T. elongatus BP-1 ΔnirA::kat mutant, which carries a gene encoding a thermostable kanamycin nucleotidyl transferase in place of nirA_Te. In the presence of nirA_Pl, nirA defective mutants of T. elongatus BP-1 recovered the ability to grow with nitrate as the sole nitrogen source, and showed a phenotype similar to that observed in wild-type cells. The procedure could be useful to substitute other genes from T. elongatus BP-1 with the homologues from P. laminosum in order to study this particular operon. Furthermore, it may be used as a general tool to explore phenotypic changes due to the exchange of a single gene between cyanobacteria.展开更多
文摘Background Donor organ rejection continues to be a significant problem for patients receiving transplants. We therefore tested whether transferring a donor＇s major histocompatibility complex （MHC） gene to the recipient would mitigate the rejection of transplanted hearts in mice. Methods H-2K^kgene from donor mice was amplified using nested polymerase chain reaction （PCR） and ligated into a mammalian expression vector, which was then transfected into thymus ground mass cells collected from the recipients. Clones stably expressing the transgene were then injected into the recipients＇ thymus visualized using ultrasound. Control mice were administered cells previously transfected with empty vector. Following heart transplantation, cardiac activity was monitored electrocardiographically. Recipient thymus cells were tested for MHC antigenicity using flow cytometry and spleen cells were subjected to mixed lymphocyte culture tests. Finally, the transplanted hearts were sectioned, stained and examined under light microscopy. Results Southern analysis following nested PCR revealed clear expression of H-2K^ gene. Following transplantation, electrocardiosignals were detectable highly significantly longer in recipients administered thymal cells expressing donor H-2K^ than in those receiving control cells. Flow cytometric analysis using an anti-H-2K^ antibody confirmed its expression in H-2K^ treated recipients but not in control mice. Mixed lymphocyte cultures containing H-2K^ treated cells showed significantly less proliferation than those containing control cells. Hearts from control mice showed substantially greater lymphocyte infiltration than those from H-2K^ treated mice and large areas of necrosis. Conclusion Rejection of transplanted hearts can be mitigated substantially by introducing the donor＇s MHC into the recipient.
文摘Even though the nitrate assimilation operon has been extensively studied in Phormidium laminosum, some aspects still remain unclear. The genetic manipulation of this cyanobacterium is problematic that hinders the elucidation of further aspects of nitrogen metabolism. To circumvent this, Thermosynechococcus elongatus BP-1 was selected as a surrogate host and its nirA gene was substituted by the homologous gene of P. laminosum. This process, based on Long Flanking Homology Polymerase Chain Reaction and the natural competence of T. elongatus BP-1, required an intermediate T. elongatus BP-1 ΔnirA::kat mutant, which carries a gene encoding a thermostable kanamycin nucleotidyl transferase in place of nirA_Te. In the presence of nirA_Pl, nirA defective mutants of T. elongatus BP-1 recovered the ability to grow with nitrate as the sole nitrogen source, and showed a phenotype similar to that observed in wild-type cells. The procedure could be useful to substitute other genes from T. elongatus BP-1 with the homologues from P. laminosum in order to study this particular operon. Furthermore, it may be used as a general tool to explore phenotypic changes due to the exchange of a single gene between cyanobacteria.
