Chromosomal translocations induced at specified loci in human stem cells
Erika Brunet1, Deniz Simsek, Mark Tomishima
1Department of Medicine, Developmental Biology Program and Sloan-Kettering Institute Stem Cell Research Facility, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
Summary
Researchers used zinc finger nucleases (ZFNs) to create targeted chromosomal translocations in human stem cells. This new method allows the study of cancer-causing genomic rearrangements in relevant human cell types.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- Chromosomal translocations are key features of many human cancers, often originating in stem or precursor cells.
- Existing methods for studying translocation formation are limited, particularly in human cells.
Purpose of the Study:
- To develop a method for inducing and analyzing chromosomal translocations in human cells, including stem cells.
- To investigate the mechanisms and consequences of targeted translocations in relevant human cell types.
Main Methods:
- Utilized zinc finger nucleases (ZFNs) to create targeted DNA double-strand breaks (DSBs) at specific loci (PPP1R12C/p84 on chromosome 19 and IL2Rgamma on the X chromosome).
- Detected translocation breakpoint junctions (t(19;X)) using quantitative PCR, DNA sequencing, and denaturation curves in a high-throughput 96-well format.
- Applied the method to various human cell types, including embryonic stem (hES) cells and their derivatives.
Main Results:
- Successfully induced and detected chromosomal translocations (t(19;X)) in human cells, including pluripotent and multipotent cells.
- Translocations were less frequent than other DSB repair pathways like gene targeting or nonhomologous end-joining.
- The developed approach is readily applicable to primary human cells, overcoming limitations of previous labor-intensive methods.
Conclusions:
- This ZFN-based technology enables the study of targeted chromosomal translocations in human stem and precursor cells.
- Provides a valuable tool for understanding the mechanisms and phenotypic effects of genomic rearrangements in cancer development.
- Facilitates research into the analysis, prevention, and treatment of human malignancies.
More Related Videos
Related Concept Videos
01:24Methods of Nuclear Reprogramming
Nuclear reprogramming is a process of transforming one cell type into an unrelated cell type by epigenetic changes that alter the cell’s original gene expression pattern. Such epigenetic changes force cells to express a different set of genes, which play a significant role in inducing transformation into other cell types. Nuclear reprogramming offers applications in reproductive cloning for livestock propagation and regenerative medicine — developing patient-specific cells for injury repair.
02:53Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
01:32Chromatin Modification in iPS Cells
Chromatin modification alters gene expression; therefore, scientists can add histone-modifying enzymes, histone variants, and chromatin remodeling complexes to somatic cells to aid reprogramming into pluripotent stem (iPS) cells.
Compact chromatin makes reprogramming difficult. Enzymes, such as histone demethylases and acetyltransferases, are often added during reprogramming to loosen the chromatin, making the DNA more accessible to transcription factors. Molecules that inhibit histone...
Compact chromatin makes reprogramming difficult. Enzymes, such as histone demethylases and acetyltransferases, are often added during reprogramming to loosen the chromatin, making the DNA more accessible to transcription factors. Molecules that inhibit histone...