FEATURE PAGE

From Embryonic Stem Cells, a Sperm Replacement and Easier Path to Genetic Modification

      Researchers reporting in the April 27 issue of the journal Cell have devised a new and improved method for producing genetically modified animals for use in scientific research. The method relies on haploid embryonic stem cells (haESCs) instead of sperm to artificially fertilize immature egg cells. Such stem cells are similar to sperm in that they carry only genetic material from a mouse "dad."

Not only will the advance make it easier to produce genetically modified mice, but it may also enable genetic modification of animals that can't be modified by today's means. The technique might ultimately be used in assisted human reproduction for those affected by genetic disease, the researchers suggest.

"The current procedure to generate genetically modified animals is tedious and very inefficient," said Jinsong Li of the Shanghai Institute for Biological Sciences. "We thought if we can generate haploid embryonic stem cells and produce semicloned animals by simply injecting those cells into oocytes, we would be certain to get a transmission into offspring with limited breeding as half of the progeny will inherit the genetic modification."

Currently, genetically modified mice are made from embryonic stem cells carrying two copies of every gene, one from mom and one from dad. These diploid embryonic cells are injected into blastocysts early in development to produce chimeras, animals whose tissues are made up of cells with one of two genomic identities. As the modified genome is randomly incorporated into the cells that will give rise to eggs and sperm, genetic modifications have the possibility to be passed on to future generations. But it's a slow and uncertain process.

Now, Jinsong Li, Guo-Liang Xu and their colleagues have found a way to generate haploid embryonic stem cells (haESCs) that can be used in place of sperm. They produce these specialized cells by first removing the nucleus from immature eggs (oocytes) and then injecting them withsperm. This procedure produces haESCs that partially retain chemical modifications characteristic of the paternal line -- enough that they can be successfully used in place of sperm.

The researchers successfully produce live mice bearing haESC-carried genetic traits. These animals, which they call "semicloned mice," grew into fertile adults.

"By being amenable to gene manipulations and supporting transmission of genetic information to offspring, these haploid cells open new avenues for the generation of genetically modified animals," the researchers write. The next challenge is to improve the sperm-like features of the haESCs by optimizing their makeup without otherwise compromising them.

The new method might also lead to genetic modification of animals, such as monkeys, that have been off limits because they don't support the production of chimeras, Li says.

As for human reproduction, right now the haESCs are clearly not as good as sperm for the purposes of IVF, but they could someday have advantages. "A similar technique might be one day used to correct genetic disease in germ cells in humans to have a healthy baby for parents," Li said.
(Cell Press (2012, April 26). From embryonic stem cells, a sperm replacement and easier path to genetic modification. ScienceDaily. Retrieved April 29, 2012, from http://www.sciencedaily.com­/releases/2012/04/120426135234.htm) 

Earliest Life Forms' Operation Promises Therapies for Diseases

         Bacteria provide a well-known playground for scientists and the evolution of these earliest life forms has shed important perspective on potential therapies for some of the most common, deadly diseases. Researchers at Case Western Reserve University School of Medicine have now discovered that, the gas nitric oxide (NO), produced in all cells of the human body for natural purposes, plays a fundamental regulatory role in controlling bacterial function, via a signaling mechanism called S-nitrosylation (SNO), which binds NO to protein molecules. In addition, the researchers discovered a novel set of 150 genes that regulate SNO production and disruption of these genes created bacterial cell damage resembling the cell damage seen in many common human diseases. Collectively these data point to new classes of antibiotics and several new disease treatments.

                  The findings, which appear in the April 27 issue of the journal Science, are significant in that they establish a parallel between how bacteria and human cells behave, and, they shed new light on how diseases that entail the same mechanism found in the bacteria may be treated.

According to the traditional Primordial Soup Theory, the earliest forms of life, including bacteria, utilize nitrate (the fertilizer) as an energy source. Its byproduct, NO, previously thought to play no significant role, is now revealed to be important for bacterial function, as it is in humans. This discovery suggests that for billions of years, NO has served as a fundamental signaling mechanism; and important related functions have been conserved in the evolution of bacteria to man.

"The mechanism, which was known to exist in human cells, but not previously thought to occur in bacteria, controls cell function and operates very broadly," says Jonathan Stamler, MD, director, Institute for Transformative Molecular Medicine and the Robert S. and Sylvia K. Reitman Family Foundation Distinguished Chair in Cardiovascular Innovation, Case Western Reserve School of Medicine and University Hospitals Case Medical Center, and director, Harrington Discovery Institute, University Hospitals Case Medical Center.

"Because the SNO mechanism can malfunction in ways that are characteristic of many diseases, what we learn from this research is immediately applicable to the development of new antibiotics and promises new insights and treatments to common diseases, including Alzheimer's, Parkinson's, heart disease, and cancer. It's not often that researchers get a big picture view of a fundamental process important to most cellular functions."

In humans, faulty NO processing contributes to many diseases, including cancer, Alzheimer's disease, Parkinson's disease, heart failure, and asthma. SNOs then build up on proteins creating specific signatures of disease. Similarly in the bacteria, the researchers found the absence of certain genes from the newly discovered set, contributed to a build-up of SNO on cell proteins. Knowing for the first time what genes are critically related to SNO build-up gives valuable insight into these disease processes. In addition, the turning on or off of the genes is a new opportunity to counter disease.

"The system we have today to control human cell function in the heart and brain evolved a billion years ago to work in bacteria. So a process that operates in bacteria is also the cause of many diseases. This offers the obvious opportunity to create new antibiotics but also therapeutic hope for multiple diseases."

The mechanism at the heart of the research is S-nitrosylation (SNO), a cellular process in which a nitric oxide (NO)-based molecule binds with a protein to activate cell signaling and fuel specific or more general cell activity.

In the event such protein modifications go awry, forming too few or too many NO attachments, disease can result. Understanding SNO binding within bacteria provides a basis for developing new drugs to disable the errant protein attachments that may contribute to disease, Dr. Stamler says. Also, drugs that disrupt the SNO controlling proteins represent novel potential antibiotics.

What keeps nitrosylation under control in bacteria, the researchers discovered, is a group of 150 genes that is regulated by the transcription factor or protein OxyR. The genes controlled by OxyR prevent aberrant NO protein attachments from taking place and keep them from interfering with normal cell function. Specifically, the genes dictate how bacteria that breathe on an ancient substance called nitrate, which they use in place of oxygen, handle nitrosative stress, a condition that results when NO molecules bind uncontrollably with protein molecules, changing their shape and function.

Prior to this research, OxyR was thought to operate only when oxygen was present. In fact, OxyR is a "master regulator" of protein S-nitrosylation that works to alleviate nitrosative stress, the new Science study shows. Relief of nitrosative stress is being sought by many companies and investigators to treat neurologic diseases, heart disease, and cancer.

Nitrosative stress is the primordial equivalent of oxidative stress, the harmful free radical injury caused by breathing in oxygen, which damages cells and contributes to aging and disease. The 150 genes identified by the Case Western Reserve researchers help manage the protein modifications that occur in bacteria as they breathe, and help eliminate NO when necessary, to avert potential cell damage or death. Without these genes, the bacteria cells would likely succumb to nitrosative stress.

Because nitrosative stress is characteristic of many diseases, including cancer and sepsis, what researchers learn about this state in bacteria can provide new perspective on these diseases and how to treat them, Dr. Stamler says. "We may be seeing disease evolution in its earliest form."

The new research builds upon Dr. Stamler's ongoing efforts to identify diseases in which protein modifications go awry, to provide a basis for the development of disease-specific drug therapies. He and his team are actively working to determine what the 150 genes identified in this research do, to isolate the genes that pertain to human diseases and spot opportunities to develop therapies to correct genetic malfunctions. Progress has already been made. 
(Case Western Reserve University (2012, April 26). Earliest life forms' operation promises therapies for diseases. ScienceDaily. Retrieved April 29, 2012, from http://www.sciencedaily.com­/releases/2012/04/120426143806.htm )