Gene trap as a tool for genome annotation and analysis of X chromosome inactivation in human embryonic stem cells

Sujoy K. Dhara, Nissim Benvenisty*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

70 Scopus citations

Abstract

Human embryonic stem (ES) cells were suggested to be an important tool in transplantation medicine. However, they also play a major role in human genetics. Using the gene trap strategy, we have created a bank of clones with insertion mutations in human ES cells. These insertions occurred within known, predicted and unknown genes, and thus assist us in annotating the genes in the human genome. The insertions into the genome occurred in multiple chromosomes with a preference to larger chromosomes. Utilizing a clone where the integration occurred in the X chromosome, we have studied X-chromosome inactivation in human cells. We thus show that in undifferentiated female human ES cells both X chromosomes remain active and upon differentiation one chromosome undergoes inactivation. In the differentiated embryonic cells the inactivation is random, while in the extra-embryonic cells it is non-random. In addition, using a selection methodology, we demonstrate that in a minority of the cells partial inactivation and XIST expression occur even in the undifferentiated cells. We suggest that X chromosome inactivation during human embryogenesis, which coincides with differentiation, may be separated from the differentiation process. The genetic manipulation of human ES cells now opens new ways of analyzing chromosome status and gene expression in humans.

Original languageAmerican English
Pages (from-to)3995-4002
Number of pages8
JournalNucleic Acids Research
Volume32
Issue number13
DOIs
StatePublished - 2004

Bibliographical note

Funding Information:
We are grateful to Dr Philip Leder (Harvard Medical School) for sharing with us the RET construct. We thank Dr Joseph Itskovitz-Eldor (Rambam Medical Center) for kindly providing us with the human ES cells as a collaborative research. S.K.D. is a Valazi-Pikovsky Fellow. This study was partially supported by funds from the Herbert Cohn Chair, by the Israel Science Foundation (grant no. 672/02-1), and by an NIH grant.

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