User:Justin R. Zabilansky
I am a new member of OpenWetWare!
- Justin R. Zabilansky
- 389-2 Grassy Hill Rd
- Lyme, CT, USA
- Email me through OpenWetWare
I work in the Runstadler Lab at MIT. I learned about OpenWetWare from From the Professor, and I've joined because To contribute to the 20.109 wiki page.
- Year, PhD, Institute
- Year, MS, Institute
- 2015, BS Course 20 Bioengineering, Massachusetts Institute of Technology
- Module 3 Research Proposal
Creating Integrin Knockout Human Chondrocytes to Realize their Role in Differentiation
Goals: Study the role of integrins in chondrocyte differentiation by creating knockout chondrocyte cells that are lacking one integrin type and observing the expression of collagen I and II
Background: Chondrocytes have been found to express several members of the integrin family, which can serve as receptors for fibronectin (α5β1), types II and VI collagen (α1β1, α2β1, α10β1), laminin (α6β1), and vitronectin and osteopontin (αVβ3). Integrin binding stimulates intracellular signaling, which regulates gene expression and cell function1.
Hypothesis: By disrupting the alpha-5 beta-1 integrin responsible for interaction with fibronectin, we expect the chondrocytes to not dedifferentiate into fibroblasts. Secondly, by disrupting the interaction of collagen types I and II, we would expect the chondrocytes to not be able to be stabilized by collagen binding and therefore dedifferentiate into fibroblasts. As laminin is expressed during the condensation of in vitro cell cultures much like fibronectin, we expect alpha-6 beta-1 knockout cells to dedifferentiate into fibroblasts. Because vitronectin plays a role in maintaining cell homeostasis, we suspect that disrupting the binding of chondrocytes to vitronectin will lead to the dedifferentiation of chondrocytes. Also, because osteopontin plays a role in recruiting nutrients for cell survival, we believe the osteopontin integrin knockout will show decreased cell viability.
Methods: The deletion of specific alpha or beta subunit genes can be done in multiple ways which may vary in their precision and throughput ability. By using type II CRISPR/Cas9 complexes which are able to use short RNAs to direct precise cleavage of DNA by the Cas9 nuclease, the specifc integrin subunit genes can be excised from a cell strains genome creating integrin knocked out cell lines. Also, site directed mutagenesis techniques can be used to inactivate the various subunit combinations in order to create cell lines with different integrins that are defective. The various cell lines with integrin genes knocked out will then be suspended in alginate gels containing all of the substrates for the different integrins (fibronectin, types II and IV collagen, laminin, vitronectin, and osteopontin). Cells from each culture will then be assessed for collagen type I and II expression using qPCR and ELISA techniques.
References: 1. http://iospress.metapress.com/content/rk900yld34r8l9m7/ 2. http://web.mit.edu/newsoffice/2013/editing-the-genome-with-high-precision-0103.html 3. http://www.ncbi.nlm.nih.gov/pubmed/23287718 4. http://www.ncbi.nlm.nih.gov/pubmed/9378775
- Virology - Currently researching the evolution of the Avian Influenza virus and tracking the species through which it propagates
- Stem Cells
- Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4.
- JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56.
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