Event Title

Development of a Cochlear Specific Cre Recombinase Expression Vector For Targeted Gene Inactivation

Location

CSU Ballroom

Start Date

21-4-2014 10:00 AM

End Date

21-4-2014 11:30 AM

Student's Major

Biological Sciences

Student's College

Science, Engineering and Technology

Mentor's Name

David Sharlin

Mentor's Email Address

david.sharlin@mnsu.edu

Mentor's Department

Biological Sciences

Mentor's College

Science, Engineering and Technology

Description

Low thyroid hormone production during a critical period of development delays remodeling process within cochlea which results in permanent auditory deficits. Unfortunately, there is large gap in understanding the molecular mechanisms that thyroid hormone regulates to control cochlear development. This project is developing an in vivo molecular tool that allows targeted gene inactivation in the greater epithelial ridge; a transient cochlear structure that is remodeled under the influence of thyroid hormone. The tectorin-alpha (TectA) gene is produced almost exclusively by cells of the GER. Considering this restricted expression pattern of TectA and our desire to generate a molecular tool for gene inactivation, we are developing a DNA construct that expresses Cre recombinase under the control of the TectA gene promoter. A 6kb TectA promoter fragment that flanks 6kb upstream of the TectA translational start codon is identified and being amplified using the PCR with appropriate primers, C57B1/6J genomic mouse DNA as a template, and a high fidelity PCR enzyme to reduce the introduction of unwanted mutations. After successfully amplification and cloning of the promoter fragment, restriction enzyme digests will be used to release the promoter region that will be subcloned, in frame, to a cre recombinase cDNA cassette. Commercial DNA sequencing will verify the final TectA promoter-Cre recombinase plasmid. We anticipate that in vitro testing will demonstrate Cre expression localized to GER cells and a future transgenic mouse line generated from the vector will be a useful molecular tool for investigating the role of candidate thyroid hormone receptor targets genes.

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Apr 21st, 10:00 AM Apr 21st, 11:30 AM

Development of a Cochlear Specific Cre Recombinase Expression Vector For Targeted Gene Inactivation

CSU Ballroom

Low thyroid hormone production during a critical period of development delays remodeling process within cochlea which results in permanent auditory deficits. Unfortunately, there is large gap in understanding the molecular mechanisms that thyroid hormone regulates to control cochlear development. This project is developing an in vivo molecular tool that allows targeted gene inactivation in the greater epithelial ridge; a transient cochlear structure that is remodeled under the influence of thyroid hormone. The tectorin-alpha (TectA) gene is produced almost exclusively by cells of the GER. Considering this restricted expression pattern of TectA and our desire to generate a molecular tool for gene inactivation, we are developing a DNA construct that expresses Cre recombinase under the control of the TectA gene promoter. A 6kb TectA promoter fragment that flanks 6kb upstream of the TectA translational start codon is identified and being amplified using the PCR with appropriate primers, C57B1/6J genomic mouse DNA as a template, and a high fidelity PCR enzyme to reduce the introduction of unwanted mutations. After successfully amplification and cloning of the promoter fragment, restriction enzyme digests will be used to release the promoter region that will be subcloned, in frame, to a cre recombinase cDNA cassette. Commercial DNA sequencing will verify the final TectA promoter-Cre recombinase plasmid. We anticipate that in vitro testing will demonstrate Cre expression localized to GER cells and a future transgenic mouse line generated from the vector will be a useful molecular tool for investigating the role of candidate thyroid hormone receptor targets genes.

Recommended Citation

Zawed, Abrar. "Development of a Cochlear Specific Cre Recombinase Expression Vector For Targeted Gene Inactivation." Undergraduate Research Symposium, Mankato, MN, April 21, 2014.
http://cornerstone.lib.mnsu.edu/urs/2014/poster_session_A/32