Files and links (1)
PDFOpen Access (License Unspecified), Open Access
Thesis
Development of RNAi-based genetic tools to silence MKP3 expression in a cell-specific manner
Master of Science (M.S.), Drexel University
Apr 2016
DOI:
https://doi.org/10.17918/etd-7168
Abstract
Drug addiction is believed to occur, in part, as a result of maladaptive changes in dopaminergic pathways. The extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein (MAP) kinase signaling pathway is implicated in altering dopaminergic signaling. Previous work demonstrated that MAP kinase phosphatase-3 (MKP3), a negative regulator of the ERK1/2 pathway, modulates dopamine release by altering dopamine transporter surface expression and voltage-gated calcium channel expression. Most pharmacological and genetic tools lack the ability to modulate intracellular signaling pathways in vivo in a cell-specific manner. Development of such cell-specific genetic tools can be used to study the role of long term plasticity changes in pathologies such as drug addiction. The goal of this project was to develop a cell-specific RNAi-based genetic tool to target and alter MKP3 expression in vivo. Cre recombinase-dependent expression of a microRNA-30-based shRNA vector is used to silence MKP3 expression. Cre recombinase reorients a transcript of interest and only expresses it in cells containing Cre. An inverted shRNA construct is flanked by incompatible lox P sites, and Cre recombinase reverses the orientation of the construct to enable cell-specific MKP3 silencing in vivo. This study tested 4 shRNAs that target different regions of the MKP3 reading frame to determine which shRNA induces the most effective MKP3 silencing in vitro. shRNAs were first cloned into DNA vectors that contain incompatible lox P sites and a promoter needed for expression of the shRNA. Proper cloning of shRNAs was verified with agarose gel analysis and DNA sequencing. After optimizing transfection conditions, in vitro studies showed that only 2 shRNAs significantly silenced MKP3 expression; only 1 of those shRNAs showed good red fluorescent protein (RFP) expression. Since these shRNAs were oriented in the forward direction, adding Cre to the shRNA samples reversed their orientation and prevented their expression, which was observed through reduced RFP expression and reversal of the MKP3 silencing effects seen without Cre. Thus both MKP3 silencing and RFP expression are Cre dependent. Future in vivo studies will assess MKP3's role in cocaine addiction by using the most effective shRNA to silence MKP3 expression only in dopaminergic neurons that contain Cre.
Metrics
18 File views/ downloads
23 Record Views
Details
- Title
- Development of RNAi-based genetic tools to silence MKP3 expression in a cell-specific manner
- Creators
- Sayani Patra - DU
- Contributors
- Ole V. Mortensen (Advisor) - Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- College of Medicine; Pharmacology and Physiology; Drexel University
- Other Identifier
- 7168; 991014632348804721