We describe here a resource of mouse embryonic stem (ES) cell lines that contain gene trap insertions capable of post-insertional modification. We demonstrate Floxin technology for efficient targeted modification of gene trap alleles. Loss-of-function gene trap mutations created with the pGTLxf and pGTLxr vectors are reverted and new DNA sequences inserted into the locus using Cre recombinase and a shuttle vector, pFloxin. As proof-of-principle, we used this strategy to create targeted modification of several gene trap alleles. pFloxin-contained DNA constructs are efficiently and precisely inserted, and are regulated by endogenous promoters. Possible applications include the generation of point mutations, humanized alleles, tagged alleles and insertion of nonhomologous constructs such as fluorescent reporters. The resource contains ES cell lines with compatible gene traps in more than 4,500 genes, enabling the high-throughput modification of many genes in mouse ES cells.
Centrosomes and their component centrioles represent the principal microtubule organizing centers of animal cells. Here we show that the gene underlying Orofaciodigital Syndrome 1, Ofd1, is a component of the distal centriole that controls centriole length. In the absence of Ofd1 , distal regions of centrioles, but not procentrioles, elongate abnormally. These long centrioles are structurally similar to normal centrioles, but contain destabilized microtubules with abnormal post-translational modifications. Ofd1 is also important for centriole distal appendage formation and centriolar recruitment of the intraflagellar transport protein Ift88. To model OFD1 Syndrome in embryonic stem cells, we replaced the Ofd1 gene with missense alleles from human OFD1 patients. Distinct disease-associated mutations cause different degrees of excessive or decreased centriole elongation, all of which are associated with diminished ciliogenesis. Our results indicate that Ofd1 acts at the distal centriole to build distal appendages, recruit Ift88, and stabilize centriolar microtubules at a defined length.
|Advisor:||Reiter, Jeremy F.|
|Commitee:||Evan, Gerard, Marshall, Wallace|
|School:||University of California, San Francisco|
|School Location:||United States -- California|
|Source:||DAI-B 71/11, Dissertation Abstracts International|
|Subjects:||Molecular biology, Genetics, Cellular biology|
|Keywords:||Centriole, Centrosome, Embryonic stem cells, Floxin, Gene knockout, Gene replacement, Gene targeting, Orofaciodigital syndrome type 1, Primary cilia|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be