Biologists develop efficient genetic modification of human embryonic stem cells

Featured In: Disease Research

By EurekAlert Thursday, January 7, 2024

Loading...

Biologists have developed an efficient way to genetically modify human embryonic stem cells. Their approach, which uses bacterial artificial chromosomes to swap in defective copies of genes, will make possible the rapid development of stem cell lines that can both serve as models for human genetic diseases and as testbeds on which to screen potential treatments, they say.

"This will help to open up the whole human embryonic stem cell field. Otherwise, there's really few efficient ways you can study genetics with them," said Yang Xu, professor of biology at the University of California, San Diego who directed the research. Xu and co-authors Hoseok Song and Sun-Ku Chung, both postdoctoral fellows in Xu's research group, describe their technique in the January 8 issue of the journal Cell Stem Cell.

Most attempts to alter the genetic makeup of the cells have proved too inefficient, Xu said. His group used bacterial artificial chromosomes, or BACs, to improve the yield.

BACs are synthesized circles of human DNA, which bacteria will replicate just like their own native chromosomes. Commercially available BACs can be modified within bacterial cells to insert altered copies of specific genes. Once the modified BACs are introduced into human cells, they will sometimes pair up with a matching segment of a human chromosome and swap segments of DNA, a process called homologous recombination.

The advantage of using BACS to alter the genetic code in human cells comes from the long flanking sequences on either side of the modified gene, which increases the chance that the BAC with line up with native DNA in position for a swap. Other genetic approaches have been limited by shorter segments of DNA.

Using BACs, the team was able to substitute modified genes in 20 percent of treated cells. Standard methods of genetic modification typically achieve modification in fewer than one percent of cells, Xu said.

His group successfully transferred a defective copy of the gene p53, which suppresses cancer, into a human embryonic stem cell line. By repeating the process in a second round, they developed a line of cells in which both copies of the genes were disrupted.

They also report success with a different gene, ATM, which when mutated in humans causes Ataxia-telangiectasia, a disease characterized by a host of systemic defects including increased cancer risk, degeneration of specific types of brain cells and degraded telomeres, the protective caps at the end of each chromosome.

Genetically engineered mice with two bad copies of the ATM gene share some of these traits with human patients, but not all. Neurons don't degenerate in ATM mice, for example, and the telomeres are long. "If you want to study accelerated shortening of telomeres, you can't do it in the mouse. You can only do it in human cells," Xu said.

Those differences propelled Xu's group to develop human cell lines instead, with the hope that some of the processes that go wrong in human patients could be studied in the lab. Already, they have demonstrated that their ATM-deficient embryonic stem cell line has damaged telomeres. Other characteristics, such as the degeneration of specific types of neurons, will be the subject of future experiments, Xu said.

The authors say their approach can easily be adapted to modify other human genes within other stem cells lines. For their initial work, Xu's group used a cell line that easily forms new colonies from single cells, but they also repeated the procedure in a cell line called H9, which has proved difficult to manipulate.

Because H9 was among the few cells lines approved for use by researchers funded by the federal government before new lines began to be approved in mid-December 2009, many researchers already have considerable experience with coaxing the cells into differentiating into specific types of tissues, for example, which would make the ability to genetically modify them particularly valuable.

SOURCE

Join the Discussion
Rate Article: Average 0 out of 5
register or log in to comment on this article!

0 Comments

Add Comment

Text Only 2000 character limit

Page 1 of 1

Research Exchange

Calibration Free Analysis to Measure the Concentration of Active Proteins

Nov 23 2009

An SPR-based method, Calibration Free Concentration Analysis can be used to accurately determine the concentration of active protein in a sample, relating to the specific binding activity of the protein, and without the need for a standard.

Advances in EMCCD Technology: Making Imaging Less Arbitrary

Nov 16 2009

Recent advances in EMCCD technology have solved the problem of non-standardized measurement units by using the photoelectron to standardize imaging experiments.

10 Tips for Successful Sample Concentration and Buffer Exchange

Nov 6 2009

Centrifugal devices with ultrafiltration (UF) membrane can solve common problems researchers face when working with proteins.

Meeting the Challenges of Long-term Time-lapse Imaging

Oct 26 2009

Recent technological developments in the sequence capture of cellular events through a light microscope can be combined to image multiple independent experiments automatically, with many advantages.

Advantages of Monolithic Laser Combiner Technology in Confocal Microscopy Systems

Jan 6

Fluorescence microscopy techniques require a reliable light source at the desired wavelength or wavelengths, with minimal downtime for maintenance and alignment. Lasers are a popular light source, although the alignment and upkeep of laser combiners is a time-consuming prospect for many users.

Size-Exclusion Chromatography for Purification of Biomolecules

Dec 2 2009

Size-exclusion chromatography (SEC) is a popular method to separate biomolecules based on their size. Primarily, it is applied to the separation of biopolymers such as proteins and nucleic acids, i.e. water-soluble polymers.

Improving Separation During Electrophoresis

Dec 2 2009

SeparateIT gels represent a novel gel matrix for DNA electrophoresis. Gel polymers are arranged in a conceptually different way, in accordance with a new theoretical model of gel electrophoresis.

Improving Quality of ELISA

Dec 2 2009

Using ready-to-use ELISA kits from manufacturers is easy and convenient. Sometimes however, home-made ELISA is required because there is no kit available with the right antibodies or the characteristics of the available kits such as their limits of detection are not appropriate.

Attention Deficit & Hyperactivity in a Drosophila Memory Mutant

Attention Deficit & Hyperactivity in a Drosophila Memory Mutant

Nov 9 2009

Action selection is modulated by external stimuli either directly or via memory retrieval. In a constantly changing environment, animals have evolved attention-like processes to effectively filter the incoming sensory stream. These attention-like processes, in turn, are modulated by memory. The neurobiological nature of how attention, action selection and memory are inter-connected is unknown. We describe here new phenotypes of the memory mutant radish in the fruit fly Drosophila.

Mechanisms of plasticity in simple taxis behaviors in Drosophila

Nov 8 2009

Like the proverbial moth drawn to the candle flame, the fruit fly Drosophila also stereotypically approaches light sources. This positive phototaxis is the archetypal example of hard-wired input-output behaviors.

Live cell imaging of mutant and wild-type GABA-A receptor trafficking using a novel reporter protein

Nov 8 2009

Efficient signaling in the brain requires precise regulation and targeting of cell surface ion-channels. Mutations in these channels associated with inherited diseases can cause improper targeting and reduced surface expression.

ADP-Glo™: An Ideal Approach to Monitor the Activity of Protein Kinases and Beyond

Oct 16 2009

Because of its versatility (alltypesofsubstrates), robustness (Z’>0.8), rapidperformance, and its ease of use, the luminescence based Kinase Glo® assay platform has gained wide acceptance in many drug screening programs for protein kinase inhibitors.

The engineering of brown fat.

14 hours ago

The developmental origins of brown adipose tissue and white adipose tissue are distinct, with brown adipocytes being derived from muscle precursors. PR domain containing 16, together with C/EBPbeta, forms a lineage-switching transcriptional complex which promotes brown fat...

Rapamycin: The Cure for all that Ails.

14 hours ago

Target of rapamycin (TOR) signaling stimulates cell growth by regulating protein synthesis in response to a variety of stimuli in a wide range of species and is inhibited by rapamycin, a naturally occurring antifungal compound produced by bacteria and discovered on Easter...

Direct activation of protein kinases by ubiquitin.

14 hours ago

The inhibitor of kB kinase (IKK) complex, a critical positive regulator of nuclear factor kappaB (NF-kappaB) signaling and immune function, can be activated in vitro by polyubiquitin chains. The modification of signaling intermediates by polyubiquitin and their association with...

Identity crisis when telomeres left unprotected.

14 hours ago

Loss of shelterin components TRF2 or POT1a-TPP1 complex from the chromosome end triggers DNA damage response (DDR) and aberrant DNA repair events. In a recent Nature paper, Chang and colleagues reported that the DNA repair protein Mre11 contributes to multiple events at the...

Prokariotic Cell Collection in Denmark

Nov 6 2009

I would like to know about a prokariotic cell collection in Denmark. Is there a cell bank in this country? I need a Lactobacillus strain for a fermentation assay and this information about the bank is very helpful for me.

Request for Entries

Oct 16 2009

Ask the Experts is your chance to get the answers to questions on applications, materials, methods, processes, and technologies. Email you question to bst_web@advantagemedia.com, and the editors of Bioscience Technology will find an appropriate expert to answer it. Watch this space in the future to see the questions your colleagues are posting.

STAY INFORMED: SUBSCRIBE TO

Magazine and E-mail Newsletters

Loading...
E-mail:   

MULTIMEDIA

Video:

Neuroscience Diseases of The Brain and How The Mind Emerges

Neuroscience Diseases of The Brain and How The Mind Emerges

Nov 8 2009

Dennis Choi, director of Emory Universitys Neuroscience Center, is renowned for his groundbreaking research on brain and spinal cord injury.

Podcasts:

Allen Institute for Brain Research

Allen Institute for Brain Research

Oct 14 2009

Discussed in this interview are both the mouse brain project and the human cortex project with an emphasis on the importance of these projects to neuroscience research.

Information: