Manipulating mouse genes to order, CRISPR or old-school

A comparison of the new vs old ways of doing gene targeting in mice. Plus, the dazzling array of options available to investigators.

Just a follow-up to last week’s announcement from the Emory Transgenic Mouse and Gene Targeting core that they are offering CRISPR/Cas9 gene editing for mice. Using CRISPR/Cas9 to produce genetically altered mice is a

Knockout_mice
Gene targeting – the 20th century way

substantial advance over the old way of doing knockouts and other manipulations (which itself won a Nobel Prize in 2007), mainly because it’s faster and easier.

To appreciate the difference, consider that the old way involves introducing DNA into mouse embryonic stem cells, and then selecting for the rare cells that take up and incorporate the DNA in the right way. Then the ES cells have to be injected into a blastocyst, followed by mouse breeding to “go germline.”

With CRISPR/Cas9, it’s possible to inject pieces of RNA that target the desired genetic changes, straight into a one-cell stage mouse embryo. Not every embryo has all the right changes, but the frequency is high enough to inject and screen. As this review explains, it’s possible to introduce mutations into three genes at once and get mice quickly, rather than make each one separately and then breed the mice together, which can take many months.

Also, because of the need for drug selection, the targeting construct in old-school gene targeting has to be a blunt instrument. That can make it hard to make subtle changes to a gene — like introduce point mutations corresponding to natural variations linked with human disease — without taking a sledgehammer to the entire gene locus. CRISPR/Cas9 takes care of that problem.

Despite the advantages of this technology, three things to keep in mind:

*Many genetically altered mice are already available “off the shelf” as part of the International Knockout Mouse/Mouse Phenotyping Consortium.

*Emory’s Mouse Core has been working with the company Ingenious Gene Targeting, and has been out-sourcing some of the tedious aspects of old-school gene targeting in mice to Ingenious, starting last year. Technicians there can generate a dazzling array of conditional knockouts. If you want your favorite gene to flip around and produce a fluorescent protein when you give the mice an antibiotic, but only in some cells — Ingenious can do that. Old school is actually still the way to go for fancy stuff like this.

*Jackson Labs in Maine also works with Emory, offering similar services, and offers a guarantee. Continue reading “Manipulating mouse genes to order, CRISPR or old-school”

Three-stage delivery for platinum-based “cluster bombs” against cancer

Scientists have devised a triple-stage “cluster bomb” system for delivering the chemotherapy drug cisplatin, via tiny nanoparticles designed to break up when they reach a tumor. They have not been tested in humans, although other approaches to packaging cisplatin are in clinical trials.

Scientists have devised a triple-stage “cluster bomb” system for delivering the chemotherapy drug cisplatin, via tiny nanoparticles designed to break up when they reach a tumor.

Details of the particles’ design and their potency against cancer in mice are described this week in PNAS Early Edition. They have not been tested in humans, although similar ways of packaging cisplatin have been in clinical trials. Anticancer cluster bombs

What makes these particles distinctive is that they start out relatively large — 100 nanometers wide – to enable smooth transport into the tumor through leaky blood vessels. Then, in acidic conditions found close to tumors, the particles discharge “bomblets” just 5 nanometers in size.

Inside tumor cells, a second chemical step activates the platinum-based cisplatin, which kills by crosslinking and damaging DNA. Doctors have used cisplatin to fight several types of cancer for decades, but toxic side effects – to the kidneys, nerves and inner ear — can limit its effectiveness.

The PNAS paper is the result of a collaboration between a team led by professor Jun Wang, PhD at the University of Science and Technology of China, and researchers led by professor Shuming Nie, PhD in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory. Nie is a member of the Discovery and Developmental Therapeutics research program at Winship Cancer Institute of Emory University. The lead authors are graduate student Hong-Jun Li and postdoctoral fellows Jinzhi Du, PhD and Xiao-Jiao Du, PhD.

“The negative side effects of cisplatin are a long-standing limitation for conventional chemotherapy,” says Jinzhi Du. “In our study, the delivery system was able to improve tumor penetration to reach more cancer cells, as well as release the drugs specifically inside cancer cells through their size-transition property.”

The researchers showed that their nanoparticles could enhance cisplatin drug accumulation in tumor tissues. When mice bearing human pancreatic tumors were given the same doses of free cisplatin or cisplatin clothed in pH-sensitive nanoparticles, the level of platinum in tumor tissues was seven times higher with the nanoparticles. This suggests the possibility that nanoparticle delivery could restrain the toxic side effects of cisplatin during cancer treatment. Continue reading “Three-stage delivery for platinum-based “cluster bombs” against cancer”

Rare inherited musculoskeletal disorder illustrates broader themes

Studying rare mutations can lead to blockbuster drugs — PCSK9 inhibitors are a prominent example. In the case of FHL1, it hasn’t happened yet, but the diverse set of effects FHL1 mutations have on skeletal and cardiac muscle are intriguing.

More than fifteen years ago, Emory geneticist William Wilcox was a visiting professor in Montevideo, Uruguay. There he worked with local doctors, led by Roberto Quadrelli, to study a family whose male members appeared to have an X-linked inherited disorder involving heart disease and musculoskeletal deformities.

In March 2016, Wilcox and his colleagues reported in Circulation: Cardiovascular Genetics that they had identified the genetic mutation responsible for the disorder, called “Uruguay syndrome.” His former postdoc Yuan Xue, now a lab director at Fulgent Diagnostics and a course instructor in Emory’s genetics counseling program, was the lead author.

Wilcox_William_Genetics_22
William Wilcox, MD, PhD

“It took many years and advances in technology to move the molecular definition from localization on the X chromosome to a specific mutation,” Wilcox says.

Still, with current DNA sequencing technology, this kind of investigation and genetic discovery takes place all the time. Why focus on this particular paper or family?

*This gene is a big tent — Mutations in FHL1, the gene that is mutated in the Uruguayan family, are responsible for several types of inherited muscle disorders, which differ depending on the precise mutation. In 2013, an international workshop summarized current knowledge on this family of diseases.

Some forms of FHL1 mutation are more severe, such as reducing body myopathy, which can have early childhood onset leading to respiratory failure. Other forms are less severe. While some men in the Uruguayan family died early from heart disease, the man who Wilcox helped treat is now teaching high school and his hypertrophic cardiomyopathy is stable on a beta blocker.

“Studying a sample of his muscle proved that we had the right gene and some of what the mutation does,” Wilcox says.

*Studying rare mutations can lead to blockbuster drugs – The discovery of potent yet expensive cholesterol-lowering PCSK9 inhibitors, which grew out of the study of familial hypercholesterolemia, is a prominent example.

FHL1 regulates muscle growth by interacting with several other proteins. Probing its function may yield insights with implications for the treatment of muscular dystrophies and possibly for athletes. As NPR’s Jon Hamilton explains, the development of myostatin inhibitors, intended to help people with muscle-wasting diseases, has led to concern about them becoming the next generation of performance-enhancing drugs. Continue reading “Rare inherited musculoskeletal disorder illustrates broader themes”

Measuring microbiome disruption

The diversity of our internal gardens could be measured by a “microbiome disruption index”. Paper from Colleen Kraft and Emory + CDC colleagues. The illustration comes from a 2014 Emory Medicine article on fecal microbial transplant.

How should doctors measure how messed up someone’s intestinal microbiome is?

This is the topic of a recent paper in American Journal of Infection Control from Colleen Kraft and colleagues from Emory and the Centers for Disease Control and Prevention. The corresponding author is epidemiologist Alison Laufer Halpin at the CDC.

A “microbiome disruption index” could inform decisions on antibiotic stewardship, where a patient should be treated or interventions such as fecal microbial transplant (link to 2014 Emory Medicine article) or oral probiotic capsules.

What the authors are moving towards is similar to Shannon’s index, which ecologists use to measure diversity of species. Another way to think about it is like the Gini coefficient, a measure of economic inequality in a country. If there are many kinds of bacteria living in someone’s body, the disruption index should be low. If there is just one dominant type of bacteria, the disruption index should be high.

In the paper, the authors examined samples from eight patients in a long-term acute care hospital (Wesley Woods) who had recently developed diarrhea. Using DNA sequencing, they determined what types of bacteria were present in patients’ stool. The patients’ samples were compared with those from two fecal microbial transplant donors. Continue reading “Measuring microbiome disruption”

Starvation signals control intestinal inflammation in mice

Intestinal inflammation in mice can be dampened by giving them a diet restricted in amino acids. Critical role for the amino acid sensor GCN2. Nature paper from Bali Pulendran and colleagues.

Intestinal inflammation in mice can be dampened by giving them a diet restricted in amino acids, the building blocks of proteins, researchers have found. The results were published online by Nature on Wednesday, March 16.

The findings highlight an ancient connection between nutrient availability and control of inflammation. They also suggest that a low protein diet — or drugs that mimic its effects on immune cells — could be tools for the treatment of inflammatory bowel diseases, such as Crohn’s disease or ulcerative colitis.

The research team, led by Emory Vaccine Center immunologist Bali Pulendran, discovered that mice lacking the amino acid sensor GCN2 are more sensitive to the chemical irritant DSS (dextran sodium sulfate), often used to model colitis in animals. This line of research grew out of the discovery by Pulendran and colleagues that GCN2 is pivotal for induction of immunity to the yellow fever vaccine.

“It is well known that the immune system can detect and respond to pathogens, but these results highlight its capacity to sense and adapt to environmental changes, such as nutritional starvation, which cause cellular stress,” he says.

Continue reading “Starvation signals control intestinal inflammation in mice”

Two Emory connections for Zika brain research

Two Emory connections to a recent Cell Stem Cell paper, showing how Zika attacks cortical progenitor cells.

Emory researchers were part of a recent advance in understanding how the Zika virus harms the developing brain. The research was published March 4 in Cell Stem Cell. 

Emory geneticist Peng Jin and his colleagues were part of a rapidly assembled research team, including scientists from Johns Hopkins and Florida State University, that showed the Zika virus can infect neural progenitor cells critical for brain development.

The research suggests a potential explanation for the cases of microcephaly seen in Latin America during the Zika outbreak. While it does not prove the direct link between Zika and microcephaly, it is a first step that shows where the virus may be doing the most damage.

The team showed that the Zika virus infects a type of neural stem cell that gives rise to the brain’s cerebral cortex. The researchers used neural progenitor cells, formed from induced pluripotent stem cells (iPSCs). The scientists showed that the virus infects neural progenitor cells more readily than iPSCs or immature neurons.

Zhexing Wen, PhD

The role of Jin’s lab was to analyze how the patterns of gene activity in neuronal cells were altered by Zika infection. Jin reports the team is continuing to examine the differences between the effects of Zika and other related viruses such as dengue and West Nile.

In addition, Lab Land recently learned that one of the scientists from Johns Hopkins, Zhexing Wen, was recruited to Emory as faculty and will start in June. His research won’t be all about Zika — in Guo-li Ming’s lab, Wen gained experience using iPSCs to model complex brain disorders such as schizophrenia. Continue reading “Two Emory connections for Zika brain research”

Silence away, HD investigators!

Adult mice don’t need the gene that, when mutated in humans, causes the inherited neurodegenerative disorder Huntington’s disease. The finding suggests that treatment strategies for Huntington’s that aim to shut off the huntingtin gene in adults — now in early clinical stages — could be safe.

Adult mice don’t need the gene that, when mutated in humans, causes the inherited neurodegenerative disorder Huntington’s disease. The finding suggests that treatment strategies for Huntington’s that aim to shut off the huntingtin gene in adults — now in early clinical stages — could be safe.

The results were published Monday, March 7 in PNAS.

How HD gene silencing is supposed to work. The Emory study didn’t test this approach directly, but the Emory study has implications for what types of side effects HD gene silencing may have in humans. Image from HDBuzz.net via Creative Commons.

Huntington’s disease is caused by a gene encoding a toxic protein (mutant huntingtin) that causes brain cells to die. Symptoms commonly appear in mid-life and include uncontrolled movements, balance problems, mood swings and cognitive decline. A juvenile form of Huntington’s disease also can appear during the teenage years.

Researchers led by Xiao-Jiang Li, MD, PhD and Shihua Li, MD, at Emory University School of Medicine, used genetically engineered mice in which the huntingtin gene can be deleted, triggered only when the mice are given the drug tamoxifen. Note: these mice don’t produce toxic mutant huntingtin protein.

When the huntingtin gene is deleted at an age older than four months, these mice appeared to stay healthy, despite having lost their huntingtin genes in cells all over their bodies. They maintained their body weight and could complete tests of movement and grip strength as well as control mice. In contrast with adults, engineered mice younger than four months old whose huntingtin gene was deleted developed lethal pancreatitis.

Continue reading “Silence away, HD investigators!”

Lung cancer cells go amoeboid

When lung cancer cells get more dangerous, they become more amoeboid

Cancer biologists Jessica Konen and Scott Wilkinson, in Adam Marcus’ lab, recently published a paper on the function of LKB1, a gene that is often mutated in lung cancer cells. [Number three behind K-ras and p53.]

Amoeboid
Mesenchymal shape is defined as having a length more than twice the width. Amoeboid looks more like the cell on the right: rounded up. Thanks to Jessica Konen for photo.

Konen and Marcus were featured in a prize-winning video that our team produced last year, which discusses how they developed a technique for isolating “leader cells” — lung cancer cells that migrate and invade more quickly — from a large group and studying those cells’ properties more intensively.

The Molecular Biology of the Cell paper covers a related topic: how LKB1 mutation affects cell shape. In particular, losing LKB1 converts lung cancer cells from a “mesenchymal” morphology to an “amoeboid” morphology.  Continue reading “Lung cancer cells go amoeboid”

Mulligan WABE interview on Ebola vaccine research

A recent WABE “Closer Look” interview with Mark Mulligan, executive director of the Emory Vaccine Center’s Hope Clinic, covers a lot of ground. Several candidate Ebola vaccines mentioned.

A recent WABE “Closer Look” interview with Mark Mulligan, executive director of the Emory Vaccine Center’s Hope Clinic, covers a lot of ground. It starts off with a segment — also aired on Marketplace — from reporter Michell Eloy, who visited the Hope Clinic’s lab. We hear a machine processing blood samples from a study testing an experimental Ebola vaccine and a roundup of Ebola vaccine developments.

We also hear from Carl Davis, postdoc in Rafi Ahmed’s lab, who is part of the DARPA-funded team research project studying the utility of antibodies from Ebola survivors. [Other recent news on this topic from The Scientist.]

Then, reporters Rose Scott and Jim Burress discuss several different Ebola vaccines with Mulligan. One is based on chimpanzee adenovirus, was tested at the Hope Clinic and elsewhere in the USA and the UK, and then in Liberia. While this vaccine was safe and it appears to stimulate the immune system appropriately, the outbreak fizzled out (a good thing!) before it was possible to tell if the vaccine protected people from Ebola infection. Continue reading “Mulligan WABE interview on Ebola vaccine research”

Beyond CF – potential byproducts of precision medicine

Other things can impair the functioning of the CFTR protein besides genetic mutations. Namely, smoking.

Just a quick comment on the potential of research being conducted by Eric Sorscher, who came to Emory from University of Alabama, Birmingham in 2015 and is now a Georgia Research Alliance Eminent Scholar. While Sorscher’s lab is working on advancing new treatments for cystic fibrosis patients who currently do not benefit from available drugs, it was intriguing to learn of potential side benefits beyond cystic fibrosis.

Cystic fibrosis is caused by mutations in the CFTR gene, which encodes a protein with important functions in cells that produce mucus, sweat, saliva, tears and digestive enzymes. But other things can impair the functioning of the CFTR protein besides genetic mutations. Namely, smoking. Continue reading “Beyond CF – potential byproducts of precision medicine”