Project Summary

Genomic evolution has created the sex chromosomes in many animal families as means of regulating sexual differentiation^1-4. In mammals two copies of an X chromosome cause female sexual development, and a single X and a Y cause male sexual development⁴. In mammals a compensation mechanism has evolved to silence one copy of the X chromosome in females⁵. This process, known as X chromosome inactivation (XCI), occurs randomly in the embryo early in development creating mosaic expression of the X chromosome; some cells express genes from the maternal X chromosome while others express the parental copy^5,6. In mice X chromosome silencing is near complete, while in humans 10-20% of the genes on the X chromosome escape X chromosome inactivation⁷. In either case this creates a delicate scenario where males have only one copy of the X chromosome and females typically express genes from only one copy.

Globally, the human X chromosome contains only 4% of genes (1098), however as of March 2009, the OMIM database had greater than 10% of all known Mendelian diseases linked to the X chromosome (384/3787). Of the X-linked diseases 37% (142/384) have yet to be associated with a specific gene. There is clearly still a major need to associate the function of X-linked genes with genetic disease. Comparative analysis of the mouse and human genome sequences reveals that there is a high degree of conservation and synteny between their X chromosomes, with 90% of known genes (629/699) conserved⁸. Given the ability to functionally alter the mouse genome, the mouse is an ideal organism to generate mutant models of human X-linked disorders.

The screen is designed to capture morphological and expression pattern information at embryonic day 9.5 by combining gene trap mouse embryonic stem cell lines (mESC) and mESC/embryo aggregation technology. Gene trapped ES cells are generated by several groups globally and a central repository maintains a database of all available lines⁹. A gene trap cell line is generated by the random insertion of a trap vector into ES cells^10,11. Typically these vectors are a splice acceptor followed by a detectable marker (Beta-galactosidase) and a drug resistance marker with a poly A signal^10,11. Tetraploid aggregation^12,13 and delayed diploid aggregation¹⁴ can both be used to generate embryos that are fully derived from mESCs.

1. Large numbers of hemizygous X-linked mutant male ES cell derived embryos can be generated.
2. Gene and mutation are defined a priori (i.e., No need to do linkage analysis, chromosomal walking, or sequencing of candidate genes).
3. beta-galactosidase insertion aids in the identification of primary phenotypes and provides valuable gene expression information.
4. mESC based screen abrogates the need for establishing germ line transmission and animal breeding.