COLLEGE STATION – People smell them, thump them and eyeball their shape. But ultimately, it’s sweetness and a sense of healthy eating that lands a melon in a shopper’s cart.

Color melon flesh is full of nutrients. Plant breeders may develop even better varieties now that the melon genome with hundreds of DNA markers has been mapped . (Texas AgriLife Photo by Kathleen Phillips)

Plant breeders now have a better chance to pinpoint such traits for new varieties, because the melon genome with hundreds of DNA markers has been mapped by scientists with Texas AgriLife Research. That means tastier and healthier melons are likely for future summer picnics.

“This will help us anchor down some of the desirable genes to develop better melon varieties,” said Dr. Kevin Crosby, who completed the study with Drs. Soon O. Park and Hye Hwang. “We can identify specific genes for higher sugar content, disease resistance and even drought tolerance.”

The results are reported in the Journal of the American Society of Horticultural Sciences.

Melons are fleshy, edible cucurbits grown worldwide in a multitude of varieties. Not only are they economically important, the scientists noted, but they are a favorite among consumers internationally.

The average person in the U.S. eats about 25 pounds of melon every year, according to the Agricultural Marketing Resource Center at Iowa State University.

Scientists from France and Spain already had completed partial maps of segments of the melon DNA sequence. The Texas researchers connected those segments with new findings in their study to complete the entire melon genome map.

For the study, the Deltex ananas melon was crossed with a wild melon called TGR 1551. More than 100 of the offspring from that cross were grown in the AgriLife Research greenhouses at Weslaco, Crosby noted.

DNA was extracted from leaf tissue collected 21 days after planting. Results from these tests were integrated into partial maps created by other researchers.

Previous knowledge of melon DNA was like two sets of directions – one from Miami to Houston and the other from El Paso to Los Angeles. That would make one wonder how to get from Houston to El Paso. The study by Crosby’s group, in essence, devised the path from Miami to LA and all points between.

In addition to the complete map, the researchers located genetic markers linked to fruit sugars, ascorbic acid (vitamin C) and male sterility, which is useful for developing hybrid varieties.

The trio said the genetic map will be helpful for future studies in identifying fruit sweetness, quality, size, shape and resistance to disease.

By Kathleen Phillips – Texas A&M AgriLife

Vaseline, a known molecule from apples and a gene network encapsulated in algal gelatin are the components of a possible gene therapy which literally gets under the skin. This is what a research group in the Department of Biosystems (D-BSSE) in Basle managed to achieve.

New way to gene therapy: first implant a capsule with a particular gene under the skin, apply skin cream in order to stimulate the gene into action, which expresses an active principle which is able to escape from the capsule. (Image: P. Rüegg/ ETH Zürich)

“An apple a day keeps the doctor away”. This English proverb now has a new meaning. Marc Gitzinger from the research group of Martin Fussenegger, Professor of Biotechnology and Bioengineering Science in the Department of Biosystems (D-BSSE) in Basle, has developed a prototype for gene therapy through the skin. An important part in this is played by phloretin, an antioxidant found in apples which makes cell walls more permeable and is used in cosmetics as an anti-wrinkle agent. The researchers have presented their new therapeutic approach online in the current edition of PNAS.

Capsules and cream

The method of administration sounds very simple: first implant a capsule with a particular gene under the skin and then apply skin cream in order to stimulate the gene into action, which finally expresses an active principle which is able to escape from the capsule in a precise dose.

Fussenegger’s group has managed to do something which sounds like science fiction. The researchers have produced alginate capsules with living cells containing a specially designed genetic network. This network produces the protein SEAP. The capsules were implanted under the skin of test mice which were then coated with an ointment. This skin cream consists of commercial milk fat mixed with phloretin according to a particular formula.

And it worked. Phloretin penetrated the skin, the gel capsules and the cells contained within. As hoped for by the researchers, the antioxidant from the apples reduced the production of protein. With a large dose of phloretin in the cream, the production of SEAP could be stopped altogether.

“When developing the principle we had no particular clinical picture in mind”, emphasised the ETH professor. “We were concentrating on the route of administration through the skin”. A genetic network such as this can also be designed in such a way that when activated correctly, insulin or growth factors are produced. The researcher can imagine that certain metabolic diseases might be treatable by this method. The D-BSSE scientists have already had the method patented and hope that the pharmaceutical industry will be interested in further developing this principle.

Liver spared

This form of gene therapy has several advantages, stressed the ETH professor. It puts no strain on the liver because it has a very local action and phloretin is a molecule which can be found in everyday foodstuffs and undergoes rapid degradation in the body. Furthermore, the network can be precisely controlled and the therapy is well tolerated by the liver, adds Fussenegger. The disadvantage of orally administered therapeutic agents is that the liver, as the detoxifying organ, destroys most of the active agent before it reaches the target site.

Fussenegger is also convinced that implants are well accepted by the public. Implants can be stored in the body for a relatively long time and are easily removed after the end of therapy or in the event of complications.

This new genetic network is a typical example of progress in synthetic biology. Researchers use known and well-characterised biological components to construct artificial networks which in turn are able to produce gene products such as specific proteins. Researchers can also use certain components to make biological switches which in turn allow such systems to be switched on or off.

Reference:

Gitzinger M, Kemmer C, El-Baba MD, Weber W, Fussenegger M. Controlling Transgene Expression in Subcutaneous Implants Using a Skin Lotion Containing the Apple Metabolite Phloretin. PNAS, online publication 22 June 200. doi:10.1073/pnas.0901501106

By Peter Rüegg – ETH Zürich

Knockout results for mouse genetics

Researchers from the Wellcome Trust Sanger Institute, the University of California at Davis, and Harvard University have isolated stable, highly germline-competent embryonic stem (ES) cells from the C57BL/6N genetic background. These cells are ideal for high-throughput genetic manipulation and are the foundation for two large-scale knockout programmes that will provide targeted C57BL/6 ES cells for the scientific community.

Embryonic stem cell derived mice. The mouse on the right carries the corrected gene and has an agouti coat.

The utility of these cells was further improved by re-activating the expression of a dominant coat colour gene (agouti). This genetic change permits the visual assessment of ES cell contribution to the germline and simplifies the breeding required to generate pure inbred lines of mice.

Historically genetic manipulation of the mouse genome has been carried out mainly in embryonic stem cells derived from the 129 strain of mice. While 129 ES cells have proven to be very robust, many genetic experiments are better carried out in C57BL/6 mice, one of the best characterised inbred strains of mice and the reference strain for the mouse genome sequence.

With the completion of the mouse genome, international programmes have started that aim to knockout more than 21,000 mouse genes in C57BL/6 inbred mice. To make this feasible, robust, highly germline competent ES cells derived from C57BL/6 are required to support large-scale mouse production and phenotyping efforts that are to follow.

In their quest for suitable C57BL/6 embryonic stem cells, the team established a male cell line (JM8) from the C57BL/6N sub-strain that contained a normal complement of chromosomes and exhibited normal undifferentiated morphology when cultured on feeder cells and on gelatine-treated plates. When early passage JM8 cells were injected into blastocysts from albino mice, pups were produced that showed high coat colour chimaerism, a sex distortion in favour of males (80%) and a high proportion of chimaeras with 100% contribution to both somatic tissues and the germline.

Two further selected sub-lines, JM8.F6 (feeder-dependent) and JM8.N4 (feeder-free) were then tested for their performance in high-throughput gene targeting experiments. In these experiments the clonal germline transmission rate was reliably better than 65%. Critically, culturing JM8 cells under feeder-free conditions did not compromise their pluripotency.

Experience has shown that injection of C57BL/6N embryonic stem cells into albino (C57BL/6 Tyrc-Brd) blastocysts is a particularly favourable combination for germline transmission. A disadvantage of this combination, however, is that mice of a mixed genetic background are produced in testcrosses to albino mice (C57BL/6 Tyrc-Brd X C57BL/6N [F1]). Thus, to simply the breeding strategy required to obtain pure C57BL/6N inbred mice, the non-agouti mutation was repaired in JM8 cells.

The non-agouti mutation in C57BL/6 strains is due to an 11.8 Kb pair retrotransposon in the first intron of the agouti gene which abolishes expression. The team realised that restoring agouti function to C57BL/6N embryonic stem cells would allow visualisation of embryonic stem cell-derived mice by coat colour and permit the recovery of pure inbred mice from test crosses with C57BL6/N mice. The team therefore designed a targeting strategy to delete the retrotransposon from the locus and restore agouti gene function.

When they injected three of these ‘restored’ clones (JM8A) into albino blastocysts, all three produced chimeras with a high percentage agouti coat colour contribution and germline colonisation, with one clone, JM8A3, showing particularly favourable results. Since these JM8A lines are heterozygous for the corrected agouti allele, testcrosses with C57BL/6N mice yield embryonic stem cell–derived offspring with either agouti or black coats.

Finally, to assess the suitability of these JM8A3 cells for high-throughput gene targeting, the team performed targeting experiments and measured the clonal transmission rate of targeted clones as above. They found a clonal germline transmission rate of 80% from the injection of 11 targeted clones.

All of the cell lines produced in this project are available on request from the Knock Out Mouse Project repository and the European Conditional Mouse Mutagenesis repository.

Publication details:

Pettit S J et al. (2009) Agouti C57BL/6N embryonic stem cells – a foundation for mouse genetic resources. Nature Methods

Funding: This work was funded by the Wellcome Trust Sanger Institute, grants from the National Institute of Health and a grant from the Sixth Framework Programme of the EU.

Participating Centres:

1. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK
2. Center for Comparative Medicine, School of Veterinary medicine, University of California, California, USA
3. Brigham and Women’s Hospital, Genetics Division, Harvard Medical School, Boston, Massachusetts, USA

The Wellcome Trust Sanger Institute

La Jolla, CA—A study led by researchers at the Salk Institute for Biological Studies, has catapulted the field of regenerative medicine significantly forward, proving in principle that a human genetic disease can be cured using a combination of gene therapy and induced pluripotent stem (iPS) cell technology. The study, published in the May 31, 2009 early online edition of Nature, is a major milestone on the path from the laboratory to the clinic.

Genetically-corrected fibroblasts from Fanconi anemia patients (shown in green at the top) are reprogrammed to generate induced pluripotent stem cells, which, in turn, can be differentiated into disease-free hematopoietic progenitors, capable of producing blood cells in vitro (bottom: Erythroid colonies.) Image: Courtesy of Dr. Juan-Carlos Belmonte, Salk Institute for Biological Studies.

“It’s been ten years since human stem cells were first cultured in a Petri dish,” says the study’s leader Juan-Carlos Izpisúa Belmonte, Ph.D., a professor in the Gene Expression Laboratory and director of the Center of Regenerative Medicine in Barcelona (CMRB), Spain. “The hope in the field has always been that we’ll be able to correct a disease genetically and then make iPS cells that differentiate into the type of tissue where the disease is manifested and bring it to clinic.”

Although several studies have demonstrated the efficacy of the approach in mice, its feasibility in humans had not been established. The Salk study offers the first proof that this technology can work in human cells.

Belmonte’s team, working with Salk colleague Inder Verma, Ph.D., a professor in the Laboratory of Genetics, and colleagues at the CMRB, and the CIEMAT in Madrid, Spain, decided to focus on Fanconi anemia (FA), a genetic disorder responsible for a series of hematological abnormalities that impair the body’s ability to fight infection, deliver oxygen, and clot blood. Caused by mutations in one of 13 Fanconi anemia (FA) genes, the disease often leads to bone marrow failure, leukemia, and other cancers. Even after receiving bone marrow transplants to correct the hematological problems, patients remain at high risk of developing cancer and other serious health conditions.

After taking hair or skin cells from patients with Fanconi anemia, the investigators corrected the defective gene in the patients’ cells using gene therapy techniques pioneered in Verma’s laboratory. They then successfully reprogrammed the repaired cells into induced pluripotent stem (iPS) cells using a combination of transcription factors, OCT4, SOX2, KLF4 and cMYC. The resulting FA-iPS cells were indistinguishable from human embryonic stem cells and iPS cells generated from healthy donors.

Since bone marrow failure as a result of the progressive decline in the numbers of functional hematopoietic stem cells is the most prominent feature of Fanconi anemia, the researchers then tested whether patient-specific iPS cells could be used as a source for transplantable hematopoietic stem cells. They found that FA-iPS cells readily differentiated into hematopoietic progenitor cells primed to differentiate into healthy blood cells.

“We haven’t cured a human being, but we have cured a cell,” Belmonte explains. “In theory we could transplant it into a human and cure the disease.”

Although hurdles still loom before that theory can become practice-in particular, preventing the reprogrammed cells from inducing tumors-in coming months Belmonte and Verma will be exploring ways to overcome that and other obstacles. In April 2009, they received a $6.6 million from the California Institute Regenerative Medicine (CIRM) to pursue research aimed at translating basic science into clinical cures.

“If we can demonstrate that a combined iPS-gene therapy approach works in humans, then there is no limit to what we can do,” says Verma.

For information on the commercialization of this technology, please contact Dave Odelson at 858.453.4100, x 1223 (dodelson@salk.edu) in the Salk Office of Technology Management and Development.

Researchers who also contributed to the work include first author Ángel Raya, as well as Ignasi Rodríguez-Pizà, Rita Vassena, María José Barrero, Antonella Consiglio, Eduard Sleep, Federico González, Gustavo Tiscornia, Elena Garreta, Trond Aasen, and Anna Veiga of the Center for Regenerative Medicine in Barcelona, Spain; Guillermo Guenechea, Susana Navarro, Paula Río, and Juan Bueren of the Hematopoiesis and Gene Therapy Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas in Madrid, Spain; and Maria Castellà and Jordi Surrallés of the Department of Genetics and Microbiology, Universitat Autonoma de Barcelona.

About the Salk Institute for Biological Studies:
The Salk Institute for Biological Studies is one of the world’s preeminent basic research institutions, where internationally renowned faculty probe fundamental life science questions in a unique, collaborative, and creative environment. Focused on both discovery and mentoring future generations of researchers, Salk scientists make groundbreaking contributions to our understanding of cancer, aging, Alzheimer’s, diabetes, and cardiovascular disorders by studying neuroscience, genetics, cell and plant biology, and related disciplines.

Faculty achievements have been recognized with numerous honors, including Nobel Prizes and memberships in the National Academy of Sciences. Founded in 1960 by polio vaccine pioneer Jonas Salk, M.D., the Institute is an independent nonprofit organization and architectural landmark.

Source: Salk Institute for Biological Studies