Publication Date

2008-01-01

Availability

Open access

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2007-11-27

First Committee Member

Keith Webster - Committee Chair

Second Committee Member

Danuta Szczesna - Committee Member

Third Committee Member

Joy Lincoln - Committee Member

Fourth Committee Member

Wayne Balkan - Committee Member

Fifth Committee Member

James D Potter - Mentor

Sixth Committee Member

Glenn Kerrick - Outside Committee Member

Abstract

The aim of this project was to produce and study a murine homozygous knock-in model containing a fast skeletal regulatory light chain (RLC) containing a Asp49toAla point mutation. The D49A mutation is in the functional calcium binding loop of RLC, which is believed to modulate muscle contraction in striated muscle. To introduce the mutation, a reversible knock-out/knock-in system was employed. The Cre/Lox-P strategy was used to conditionally knock-in the RLC D49A mutation. The generation of the knock-in mouse was attempted with two different breeding strategies consisting of two Cre mouse lines with differential expression patterns during development. The proposed animal was never produced because the RLC knock-out recombination event introduced a splicing error resulting in a stop codon in intron 2. Extensive DNA, RNA and protein analysis as well as histological, gross morphology and muscle physiology studies obtained from the animals of the two breeding strategies lead to the identification of the splicing error. Evidence for this outcome is presented. A recommendation for a different strategy in future studies is included.

Keywords

Muscle Histology; Muscle Fiber Studies; Cre-LoxP; Mice Model; Knock-in Mice; Muscle; Contraction; Skeletal Muscle; Myosin Regulatory Chain

Share

COinS