Cecropins, first identified in silk moth (Hyalophora cecropia), are a group of antimicrobial peptides with bactericidal activity against a broad spectrum of bacteria. In this study we investigated whether (1) this group of antimicrobial peptides could exhibit bactericidal activity toward known fish bacterial pathogens and (2) expression of cecropin transgenes in transgenic medaka (Oryzias latipas) could result in increasing resistance of the transgenic fish to infection by fish bacterial pathogens. Cecropin gene construct containing silk moth preprocecropin B, procecropin B and cecropin B, and porcine cecropin P1 driven by a cytomegalovirus (CMV) promoter were transfected into chinook salmon embryonic cells (CHSE-214) by lipofection, and the resulting permanent transformants were collected. In an "inhibition zone" assay medium isolated from each transformant exhibited strong bactericidal activity toward known fish bacterial pathogens such as Pseudomonas fluorescens, Aeromonas hydrophila, and Vibrio anguillarum. The same cecropin transgene constructs were introduced into newly fertilized medaka eggs by electroporation to produce transgenic fish. About 40% to 60% of the embryos survived from electroporation, and about 5% to 11% of the surviving fish were shown to contain cecropin transgenes by polymerase chain reaction analysis of genomic DNA samples isolated from presumptive transgenic fish. These P1 transgenic fish were used as founder stocks, and following generations of successive breeding, a total of 20 F2 families of transgenic fish were established. Expression of cecropin transgenes was detected in the F2 transgenics by reverse transcriptase polymerase chain reaction analysis. Southern blot analysis of genomic DNA isolated from different F2 fish showed that cecropin transgenes were integrated into the genomes of F2 transgenic fish. To determine whether transgenic fish carrying cecropin transgenes could exhibit resistance to infection by known fish bacterial pathogens, F2 transgenic fish from different families and control fish were challenged with P. fluorescens and V. anguillarum at a 60% lethal dose. Challenge studies showed that while about 40% of the control fish were killed by both pathogens, only up to 10% of the F2 transgenic fish were killed by P. fluorescens and about 10% to 30% by V. anguillarum. These results clearly showed that the transgenic medaka carrying cecropin transgenes had acquired elevated resistance to bacterial infection.
Transgenic animals have been routinely produced by microinjecting or electroporating naked DNA into 1-cell-stage embryos or unfertilized eggs. However, these techniques are inapplicable to live-bearing fish and many crustacean species for which unfertilized or newly fertilized eggs are not readily obtainable. In the present study, replication-defective pantropic retroviral vectors carrying a reporter gene (neo(R) or beta-gal) were used to directly transform the immature ovary or testis of a live-bearing fish (Poeciliopsis lucida) and crayfish (Procambarus clarkii). The fraction of the progeny derived from these treated individuals shown to contain the neo(R) reporter gene by an assay based on polymerase chain reaction (PCR) was significant. The PCR-positive individuals were crossed with nontransgenic individuals, and about 50% of the resulting progeny carried the transgene, suggesting that the F(1) animals are germline transgenic. Integration of the transgenes was confirmed by detecting the junction fragments of the genomic DNA associated with transgene constructs. Expression of reporter genes was detected by a reverse transcription-nested PCR assay. These results showed that transgenic live-bearing fish and crustaceans could be easily produced by directly transforming the immature gonads with replication-defective pantropic retroviral vectors.
Measles virus plays an important role as an environmental factor in the pathogenesis of Paget’s disease (PD). Previous studies have shown that IL-6 is increased in the bone marrow of Paget’s patients and that measles virus nucleocapsid protein (MVNP) induces IL-6 secretion by pagetic osteoclasts. Further, IL-6 plays a critical role in the development of pagetic osteoclasts and bone lesions induced by PD, but the mechanisms regulating IL-6 production by MVNP remain unclear. Our current studies revealed that MVNP expression in osteoclast precursors down-regulated Sirt1 mRNA and protein, a negative regulator of NF-κB activity, which is a key factor for IL-6 expression. MVNP expression in NIH3T3 cells also elevated Il-6 transcription and impaired the expression of Sirt1 mRNA both under basal conditions and upon activation of the Sirt1 upstream regulator FoxO3 by LY294002 (a PI3K/AKT inhibitor). Luciferase activity assays showed that constitutively active FoxO3 abolished the repressive effect of MVNP on reporters driven by either FoxO3 response elements or the Sirt1 promoter. Further, protein stability assays revealed that FoxO3 was degraded more rapidly in MVNP-expressing cells than in control cells following the addition of cycloheximide. Similarly, co-transfection of MVNP and FoxO3 into HEK293 cells demonstrated that MVNP decreased the protein levels of over-expressed FoxO3 in a dose-dependent manner. Treatment with the proteasome inhibitor, MG132, blocked the MVNP-triggered decrease of FoxO3, and the treatment with the serine/threonine phosphatase inhibitor, Calyculin A, revealed that MVNP increased phosphorylation of FoxO3. Further, over-expression of Sirt1 or treatment with the Sirt1 activator resveratrol blocked the increase in Il-6 transcription by MVNP. Finally, resveratrol reduced the numbers of TRAP positive multi-nuclear cells in bone marrow cultures from TRAP-MVNP transgenic mice to wild type levels. These results indicate that MVNP decreases FoxO3/Sirt1 signaling to enhance the levels of IL-6, which in part mediate MVNP’s contribution to the development of Paget’s disease.
ECF-L is a novel autocrine stimulator of osteoclast (OCL) formation that enhances the effects of 1,25-(OH)2D3 and RANK ligand (RANKL) and is increased in inflammatory conditions such as rheumatoid arthritis. ECF-L acts at the later stages of OCL formation and does not increase RANKL expression. Thus, its mechanism of action is unclear. Therefore, RAW 264.7 cells and M-CSF-dependent murine bone marrow macrophage (MDBM) cells were treated with RANKL and/or with recombinant ECF-L expressed as a Fc fusion protein (ECF-L-Fc) to determine their effects on NF-kappaB, AP-1 and JNK activity, and on the expression of the adhesion molecules that have been implicated in OCL formation. These parameters were measured by semiquantitative and PCR and Western blot analysis. In addition, the role of ICAM-1 was further assessed by treating normal mouse marrow cultures with ECF-L-Fc and 10(-10) M 1,25-(OH)2D3 in the presence or absence of a blocking ICAM-1 antibody or treating marrow cultures from ICAM-1 knockout mice with ECF-L and 1,25-(OH)2D3. ECF-L-Fc by itself only modestly increased NF-kappaB binding and JNK activity in RAW 264.7 cells, which was further enhanced by RANKL. In contrast, ECF-L-Fc increased LFA-1alpha and ICAM-1 mRNA levels 1.8-fold in mouse marrow cultures, and anti-ICAM-1 almost completely inhibited OCL formation induced by 10(-10) M 1,25-(OH)2D3 and ECF-L. Furthermore, ECF-L did not increase OCL formation in marrow cultures from ICAM-1 knockout mice. Taken together, these results demonstrate that ECF-L enhances RANKL and 1,25-(OH)2D3-induced OCL formation by increasing adhesive interactions between OCL precursors through increased expression of ICAM-1 and LFA-1.
Transgenic fish have been routinely produced by microinjecting or electroporating foreign DNA into one-cell stage embryos or unfertilized eggs. While both techniques are effective in producing transgenic fish species from which unfertilized or newly fertilized eggs can be easily obtained, these techniques are not applicable to live-bearing fish and many crustacean species where unfertilized or newly fertilized eggs are not readily available. In this paper, we describe a new method of introducing foreign DNA into the live-bearing fish, Poeciliposis lucida, and crayfish, Procambarus clarkii, by directly transforming the immature ovary or testis of these animals with replication-defective pantropic retroviral vectors carrying a reporter gene (neo(R)). A significant fraction of the progeny derived from these treated animals contains the neo(R) reporter gene, determined by a PCR-based assay. The PCR-positive individuals were crossed with nontransgenic individuals, and about 50% of the resulting progeny carried the transgene, suggesting that the F(1) animals are germline transgenic. Integration of the transgenes was confirmed by detecting the junction fragments of the genomic DNA associated with transgene constructs. The expression of reporter genes was detected by reverse transcription (RT) PCR assay. These results showed that foreign genes could be reproducibly transferred into live-bearing fish and crustaceans by directly transforming the immature gonads with replication-defective pantropic retroviral vectors.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.