Details

Construction of a COL11A1 Transgene Vector Construction of a COL11A1 Transgene Vector

by Beck, Cameron McKell

Abstract (Summary)
Background: Cartilage disorders affect millions of people in the United States alone, with effects ranging from poor skeletal development and joint pain to shortened lifespan and perinatal lethality. Many of these disorders have their root in defects of collagen, type XI collagen being among the most important. A mouse model of such a type XI collagen defect is the chondrodysplasia (cho) mutant. Mice homozygous for this null mutation in the Col11a1 gene do not express the á1 chain of type XI collagen. This results in a functional knockout of type XI collagen, leading to insufficient skeletal development and perinatal lethality. Objective: 1) To construct a transgenic expression vector designed to express a human COL11A1 cDNA in a cartilage-specific manner. This transgene will be used in future studies to correct the type XI collagen defect in homozygous cho mice. 2) To place the cDNA in an in vitro expression vector to be used for in vitro transcription/translation assays. Methods and Results: Through the relatively new approach of “recombineering”, the coding sequence of a human COL11A1 cDNA was constructed from two cloned cDNA fragments. A copy of the cDNA was inserted into the pcDNA3.1 expression vector for in vitro transcription/translation assays.

Another copy of the cDNA was fused with a genomic mouse á-globin fragment to provide a polyadenylation signal. The resulting cDNA/á-globin segment was inserted into p1757, the expression vector to be used for future transgenic studies. p1757 contains a Col2a1 promoter, a â-globin splice sequence and a Col2a1 enhancer. The cDNA/á-globin segment was inserted between the splice sequence and the enhancer. With the cDNA in this expression cassette COL11A1 can be expressed in a chondrocyte-specific manner in transgenic studies of the cho mouse model.

Bibliographical Information:

Advisor:

School:Brigham Young University

School Location:USA - Utah

Source Type:Master's Thesis

Keywords:col11a1 transgene genetic construct transgenic rescue mouse human

ISBN:

Date of Publication:08/14/2006

© 2009 OpenThesis.org. All Rights Reserved.