Application of the floxin gene targeting technology reveals that Ofd1 controls centriole length

Abstract

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.