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Showing 2 results for Chimera
Samaneh Poursaeid, Mohammadreza Kalbassi, , , Hossein Baharvand,
Volume 11, Issue 4 (11-2020)
Abstract
Spermatogonial stem cells (SSCs) are unique with an important role in the transmission of genetic information to the next generation. Thus, they play an important role for the production of interspecies germ line chimeras. Therefore, the objective of this study was to produce chimera through the intraperitoneal transplantation of Caspian brown trout SSCs into newly-hatched rainbow trout. Spermatogonial cell were isolated from the testes of 8-month-old Caspian brown trout through enzymatic digestion. The spermatogonial cell suspension was enriched using differential plating technique to remove testicular somatic cells. After culturing for 48 h in L15 supplemented with 10% serum, suspended cells were collected and stained with the fluorescent membrane dye PKH26. The stained cells were intraperitoneally transplanted into triploid rainbow trout hatchlings. At 15 and 30 days after transplantation, the recipients were investigated under a fluorescent microscope. The gonads of recipients were dissected for molecular analysis at 180 days after transplantation. Transplanted spermatogonial cells migrated toward and incorporated into recipient genital ridges. The presence of the Caspian brown trout genetic material was confirmed by PCR in 41.4% of the rainbow trout testes. These results demonstrated for the first time that the interspecies spermatogonial transplantation was successful in rainbow trout and that the somatic microenvironment of the rainbow trout gonad can support the colonization and survival of intraperitoneally transplanted cells derived from a fish species belonging to a different genus. Therefore, the SSCs transplantation can be used as a tool for conservation of Caspian brown trout genetic resources.
Volume 22, Issue 2 (3-2019)
Abstract
Aims: The chimeric domain-exchanged streptokinase (SKch) between two sk genes from groups G and A streptococci (SK2aG88 and SK2bALAB49, respectively) was constructed to evaluate the role of SK-domains (α, β, γ) in α2-antipalsmin-resistance variations of SK.
Materials and Methods: In this experimental study, PCR-amplified genes of streptococci (skg, ska) were cloned into pET26b vector to produce pET26-SKG88 and pET26-SKALAB49. For domain exchange, the amplicon containing β and g domains of SK2bALAB49 was replaced for that of the SK2aG88 within pET-SK2aG88 (pET26-SKch; α2aG88β2bALABγ2bALAB). All constructs were confirmed by restriction analyses/agarose-gel electrophoresis and DNA sequencing, transformed into E.coli Rosetta, and induced by IPTG for protein expression. Proteins were purified by Ni-NTA chromatography, quantified by Bradford method, and analyzed by SDS-PAGE/Western blotting assays. The α2-antipalsmin-resistance was measured by S2251 colorimetric assay for plasminogen activation.
Findings: SDS-PAGE and western blotting results indicated the expression of proteins with the size of 47kD. At the highest concentration of α2-antiplasmin, SK2aG88 remained 80% active, whereas the SK2bALAB49 and SKch retained 55% of their activity.
Conclusion: SKch shows similar activity reduction, indicating the minor role of the α domain compared to β and g domains for α2-antipalsmin-resistance.
