Cloning and Expression of the DNA Binding Domain of FoxO from Ciona intestinalis That Contains an N-Terminal Nuclear Localization Signal
Poster Number
43
College
College of Arts and Sciences
Department
Biology
Faculty Mentor
Nicholas Grossoehme, Ph.D. and Heather Evans-Anderson, Ph.D.
Abstract
FoxO proteins are a subgroup of the Forkhead family of transcription factors. FoxO proteins are highly conserved and regulate expression of genes that control a wide variety of cellular processes, including: apoptosis, cell differentiation and proliferation, and atrophy. Ciona intestinalis is a useful model system to study developmental biology, particularly heart development, since all chordates share a conserved cardiac gene program, as well as similar cellular processes during development. Ciona FoxO (ciFoxO) protein is very similar to the FoxO1 protein in humans. In order for ciFoxO to transcriptionally regulate gene expression, it must localize to the nucleus. The major goal of this project is to add a nuclear localization signal (NLS) to an expression vector containing ciFoxO sequence that will be electroporated into Ciona embryos where it will be expressed. The N-terminal NLS will direct the exogenous ciFoxO sequence to the nucleus of cells, where it will be able to bind to target DNA sequences in the Ciona genome. The ultimate goal is to express ciFoxO constructs containing a NLS in vivo and then isolate chromatin in order to perform a ChIP-Seq assay to determine ciFoxO target genes. The ciFoxO target genes will be compared to vertebrate FoxO1 target genes to determine the level of conserved function of FoxO family members in chordates during heart development. To date, we have successfully inserted the NLS into the vector and produced dechorinated embryos; electroporation optimization is underway.
Start Date
24-4-2015 1:20 PM
End Date
24-4-2015 2:50 PM
Cloning and Expression of the DNA Binding Domain of FoxO from Ciona intestinalis That Contains an N-Terminal Nuclear Localization Signal
Richardson Ballroom
FoxO proteins are a subgroup of the Forkhead family of transcription factors. FoxO proteins are highly conserved and regulate expression of genes that control a wide variety of cellular processes, including: apoptosis, cell differentiation and proliferation, and atrophy. Ciona intestinalis is a useful model system to study developmental biology, particularly heart development, since all chordates share a conserved cardiac gene program, as well as similar cellular processes during development. Ciona FoxO (ciFoxO) protein is very similar to the FoxO1 protein in humans. In order for ciFoxO to transcriptionally regulate gene expression, it must localize to the nucleus. The major goal of this project is to add a nuclear localization signal (NLS) to an expression vector containing ciFoxO sequence that will be electroporated into Ciona embryos where it will be expressed. The N-terminal NLS will direct the exogenous ciFoxO sequence to the nucleus of cells, where it will be able to bind to target DNA sequences in the Ciona genome. The ultimate goal is to express ciFoxO constructs containing a NLS in vivo and then isolate chromatin in order to perform a ChIP-Seq assay to determine ciFoxO target genes. The ciFoxO target genes will be compared to vertebrate FoxO1 target genes to determine the level of conserved function of FoxO family members in chordates during heart development. To date, we have successfully inserted the NLS into the vector and produced dechorinated embryos; electroporation optimization is underway.
