Rupert et al., "The human XIST gene
: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus," Cell, vol.
Here, we extended this analysis showing that XIST was activated in only a few HCT116-5-aza-CdR cells, despite the XIST gene being hypomethylated.
An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells.
The investigators set out to determine if they could insert a copy of the XIST gene into the extra chromosome 21--at a very specific location so as not to disrupt any known functional genes--and thereby silence the extra chromosome without affecting other chromosomes.
Lawrence and her colleagues were able to accomplish this integration and overcome two major obstacles: the challenge of working with pluripotent stem cells, and inserting such a large amount of DNA (the XIST gene) via genomic editing.
The process normally uses the matching chromosome as a guide for sealing the breach, instead, Jiang supplied a template containing the enormous XIST gene
sandwiched between two bits of DNA that matched sequences on either side of the gap.
They did it by harnessing a naturally-occurring chromosome "off-switch", the XIST gene
. XIST normally has the job of silencing one of the two X chromosomes in female cells.
Determination of FMR1 gene promoter methylation: Analysis of methylation status of the FMR1 promoter and an internal control Xist gene
(X-inactive specific transcript) was carried out following the method of Weinhausel and Hass (23).
The murine Xist gene
was amplified from that with the forward primer, 5'-ACTGCCAGCAGCCTATACAG-3', and reverse primer, 5'-GTTGATCCTCGGGTCATTTA-3'.
In female Eutherian (placental) mammals, one copy of the X chromosome in every cell is inactivated, due to the activity of the Xist gene
. The Xist gene
is found only in Eutherians, and in no other vertebrates.
In brief, exon 1 (6 kb) and exon 6 (6 kb) of the XIST gene
were amplified by PCR and labeled with Cy3 by nick translation.
This technique tricks the male cell, which apparently calculates that it contains more than one X chromosome, into activating the Xist gene
and turning off a nearby gene that the researchers had added along with Xist, Jaenisch's group reported in the July 12, 1996 Cell.