Novel artificial chromosome gene expression system successfully developed in human embryonic stem cells

Researchers have, for the first time, generated artificial chromosomes in human embryonic stem (hES) cells containing genes which are expressed and maintained following differentiation of the hES cells into other cell types.

Human artificial chromosomes (HAC) are small, functional, genetic elements which behave like normal chromosomes within cells. They are generated when chromosomal DNA is artificially introduced into human cells. Several other methods exist to introduce new DNA into human cells, however, HAC are more successful and effective as they can accommodate larger genes and regulatory regions for long term studies of gene expression. They are also more safely introduced and maintained than DNA introduced using viral vectors.

Researchers at the WTCHG, led by Dr Zoia Monaco, are part of the team which has now successfully shown that stable HAC expressing genes were generated in hES cells. The DNA was targeted to the hES cell nucleus, and led to the assembly of HAC at a high frequency. A major finding was that the host cell genome remained intact, which differed from studies in tumour derived cells where the input DNA often became integrated in the host chromosomes. Cells containing the HAC retained the ability to form different cell types, such as neuronal cells, with the HAC maintained after differentiation.

This novel study highlighted significant differences in chromosome and genome stability between stem and tumour cells. HAC will be important as markers to screen for chromosome instability in cell types such as induced pluripotent stem (iPS) cells for patient therapeutic strategies.

 

The study was published in Human Molecular Genetics and is available to read online here:

http://hmg.oxfordjournals.org/content/early/2011/05/24/hmg.ddr144.full?sid=0be5368d-741a-46ef-a33e-209c29f444fc

For more information on Dr Monaco's research, click here.

Jun 11 Larin Monaco

 

Top left: FISH on hES metaphase spread, with 17 alpha satellite DNA (green signal), which also labels the endogenous chromosome 17, and a HAC specific probe (red signal). The chromosomes are counterstained in DAPI, blue. The HAC is identified by a yellow arrow.

Top right: HAC containing hES cells stained with antibody for an embryonic stem cells specific marker (Oct4, red signal). The nuclei are stained in DAPI, blue.

Bottom left: BetaIII-Tubulin antibody staining (red) of differentiated HAC-hES cells. The differentiated neuronal cells are positive to the antibody staining. The nuclei are stained in DAPI, blue.

Bottom right: FISH with HAC specific probe (red signal, yellow arrow) differentiated cells, stained with BetaIII-Tubulin antibody (green signal). The nuclei are stained in DAPI, blue.