A platform for research: civil engineering, architecture and urbanism
A platform for research: civil engineering, architecture and urbanism
Generation of transgenic mice for the investigation of ceramide metabolism ; Herstellung transgener Mäuse zur Untersuchung des Ceramid-Stoffwechsels
Ceramide self-association in cell membranes gives rise to formation of ceramide-rich domains, which in turn reorganize membrane proteins and affect reaction yields of signal transduction pathways. Deregulated ceramide metabolism and membrane organization has been shown in many disease pathologies. The present study aimed to generate transgenic mouse models for the enzymes acid ceramidase and acid sphingomyelinase which both modulate ceramide levels. Genetic mouse models are valuable scientific tools for studying physiological and pathological processes in vivo. In a “gain of function” model, acid ceramidase expression cassettes were introduced into the murine genome. The expression cassette comprised a CAG-promoter driving the transcription of acid ceramidase complementary DNA. Genetic components of the CAG-Asah1 expression cassette were assembled by DNA cloning techniques. Functionality of the construct was tested in vitro proving CAG-Asah1 dependent enhancement of acid ceramidase protein levels and activity. The transgene cassette was delivered to the murine genome by pronuclear injections into fertilized eggs. Transgenic offspring was identified by PCRs and three founder lines were established. The gene dosage of transgene copies in distinct founder lines was determined by quantitative PCR methods. Sphingosine levels of liver, kidney and spleen tissue homogenates were determined by mass spectrometry. The data revealed that tissues of CAG-Asah1 transgenic animals displayed significantly higher levels of the acid ceramidase reaction product in comparison to wild type tissues. The results can be explained by an enhanced catalytic activity of acid ceramidase in CAG-Asah1 animals. In conclusion, my research generated a CAG-Asah1 transgenic mouse model which may reveal important scientific findings with regard to the biological effects resulting from ceramide consumption by acid ceramidase. In an attempt to develop a conditional knockout model, the acid sphingomyelinase gene was targeted with a replacement vector. ...
Generation of transgenic mice for the investigation of ceramide metabolism ; Herstellung transgener Mäuse zur Untersuchung des Ceramid-Stoffwechsels
Ceramide self-association in cell membranes gives rise to formation of ceramide-rich domains, which in turn reorganize membrane proteins and affect reaction yields of signal transduction pathways. Deregulated ceramide metabolism and membrane organization has been shown in many disease pathologies. The present study aimed to generate transgenic mouse models for the enzymes acid ceramidase and acid sphingomyelinase which both modulate ceramide levels. Genetic mouse models are valuable scientific tools for studying physiological and pathological processes in vivo. In a “gain of function” model, acid ceramidase expression cassettes were introduced into the murine genome. The expression cassette comprised a CAG-promoter driving the transcription of acid ceramidase complementary DNA. Genetic components of the CAG-Asah1 expression cassette were assembled by DNA cloning techniques. Functionality of the construct was tested in vitro proving CAG-Asah1 dependent enhancement of acid ceramidase protein levels and activity. The transgene cassette was delivered to the murine genome by pronuclear injections into fertilized eggs. Transgenic offspring was identified by PCRs and three founder lines were established. The gene dosage of transgene copies in distinct founder lines was determined by quantitative PCR methods. Sphingosine levels of liver, kidney and spleen tissue homogenates were determined by mass spectrometry. The data revealed that tissues of CAG-Asah1 transgenic animals displayed significantly higher levels of the acid ceramidase reaction product in comparison to wild type tissues. The results can be explained by an enhanced catalytic activity of acid ceramidase in CAG-Asah1 animals. In conclusion, my research generated a CAG-Asah1 transgenic mouse model which may reveal important scientific findings with regard to the biological effects resulting from ceramide consumption by acid ceramidase. In an attempt to develop a conditional knockout model, the acid sphingomyelinase gene was targeted with a replacement vector. ...
Generation of transgenic mice for the investigation of ceramide metabolism ; Herstellung transgener Mäuse zur Untersuchung des Ceramid-Stoffwechsels
Knüwer, Martin (author) / Gulbins, Erich
2014-02-04
Theses
Electronic Resource
English
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