mouse cells

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In 2010, Sonia Vallabh watched her mom, Kamni Vallabh, die in a really horrible way.

First, her mom’s memory started to go, then she lost the ability to reason. Sonia says it was like watching someone get unplugged from the world. By the end, it was as if she was stuck between being awake and asleep. She was confused and uncomfortable all the time.

“Even when awake, was she fully or was she really? And when asleep, was she really asleep?” says Sonia.

The smart, warm, artistic Kamni – just 51 years old — was disappearing into profound dementia.

“I think until you’ve seen it, it’s hard to actually imagine what it is for a person to be alive and their body is moving around, but their brain is not there anymore,” says Eric Minikel, Sonia’s husband.

In less than a year, Sonia’s mom died.

An autopsy showed Kamni had died from something rare — a prion disease. Specifically, one called fatal familial insomnia because in some patients it steals the ability to fall asleep.

Basically, certain molecules had started clumping together in Kamni’s brain, killing her brain cells. It was all because of one tiny error in her DNA — an “A” where there was supposed to be a “G,” a single typo in a manuscript of 6 billion letters.

Sonia sent a sample of her own blood to a lab, where a test confirmed she inherited the same mutation. The finding threw the family into grief all over again.

Today, Sonia and her husband live and work in Cambridge, Mass., where they are both doctoral students in the lab of Stuart Schreiber, a Harvard professor of chemistry and chemical biology. Over the past several years, the couple has completely redirected their careers and their lives toward this single goal: to prevent prion disease from ever making Sonia sick.

A Couple’s Quest To Stop A Rare Disease Before It Takes One Of Them

Photos: Kayana Szymczak for NPR

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I think I mentioned on my main blog that I was doing some drawings for Pride Month. I’m not sure how many more I’m going to get done, so I’m uploading these now.

Happy Pride Month!

Cambridge scientists create first self-developing embryo from stem cells

The transformation of a fertilised egg into a tiny living embryo ranks among nature’s most impressive feats. Now scientists have replicated this critical step towards a new life for the first time, growing an artificial mouse embryo from stem cells in the lab.

The cells, grown outside the body in a blob of gel, were shown to morph into primitive embryos that perfectly replicated the internal structures that emerge during normal development in the womb.

The scientists let the artificial embryos develop in culture for seven days – about one third of the way through the mouse pregnancy. By this point the cells had organised into two anatomical sections that would normally go on to form the placenta and the embryonic mouse.

The mouse embryo breakthrough is not designed to grow mice or babies outside the womb, but to open a new window on the embryo’s development just prior to implantation. Photograph: Redmond Durrell / Alamy/Alamy

Scientists Catalogue “Parts List” of Brain Cell Types in a Major Appetite Center

Using Harvard-developed technology, scientists at Beth Israel Deaconess Medical Center (BIDMC) have catalogued more than 20,000 brain cells in one region of the mouse hypothalamus. The study, published in Nature Neuroscience, revealed some 50 distinct cell types, including a previously undescribed neuron type that may underlie some of the genetic risk of human obesity. This catalog of cell types marks the first time neuroscientists have established a comprehensive “parts list” for this area of the brain. The new information will allow researchers to establish which cells play what role in this region of the brain.

“A lot of functions have already been mapped to large regions of the brain; for example, we know that the hippocampus is important for memory, and we know the hypothalamus is responsible for basic functions like eating and drinking,” said lead author John N. Campbell, PhD, a postdoctoral fellow in the lab of co-corresponding author, Bradford Lowell, MD, PhD. “But we don’t know what cell types within those regions are responsible. Now with the leaps we’ve had in technology, we can profile every gene in tens of thousands of individual cells simultaneously and start to test those cell types one by one to figure out their functional roles.”

Each cell in an animal’s body carries the same genetic information. Cells take on specific roles by expressing some genes and silencing others. Drop-Seq technology – developed by study co-authors Steven McCarroll, PhD, and Evan Macosko, MD, PhD, both geneticists at Harvard Medical School – makes it possible to assess every gene expressed by individual cells. The automated process means the BIDMC researchers could profile tens of thousands of cells in the same amount of time it once took to profile about a dozen cells by hand.

Campbell and colleagues profiled more than 20,000 adult mouse brain cells in the arcuate hypothalamus (Arc) and the adjoining median eminence (ME) – a region of the brain that controls appetite and other vital functions. The cells’ gene expression profiles help scientists determine their functions.

In addition to identifying 50 new cell types, the researchers also profiled the cell types in adult mice under different feeding conditions: eating at will; high-fat diet (energy surplus); and overnight fasting (energy deficit). The technology allowed the researchers to assess how changes in energy status affected gene expression. The cell types and genes that were sensitive to these changes in energy status provide a number of new targets for obesity treatment.

“Sometimes a cell’s true identity doesn’t come out until you put it through a certain stress,” said co-corresponding author, Linus Tsai, MD, PhD, an assistant professor of medicine in the Division of Endocrinology, Diabetes and Metabolism at BIDMC. “In fasting conditions, for example, we can see whether there is further diversity within the cell types based on how they respond to important physiologic states.”

Finally, the scientists analyzed previous human genome-wide association studies (GWAS) that revealed gene variants linked to obesity. Noting which brain cell types express such obesity-related genes, the researchers implicated two novel neuron types in the genetic control of body weight.

Campbell and colleagues have posted their massive data set online, making it available to researchers around the world. The open-source information should accelerate the pace of scientific discovery and shape the research questions asked in the field of obesity research.

“The classic way of doing science is to ask questions and test hypotheses,” said Lowell, who is a professor of medicine in the Division of Endocrinology, Diabetes and Metabolism. “But the brain is so complex, we don’t even know how much we don’t know. This information fills in some of the unknowns so we can make new hypotheses. This work will lead to many discoveries that, without these data, people would never have even known to ask the question.”

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How to build a human cell atlas

Aviv Regev likes to work at the edge of what is possible. In 2011, the computational biologist was collaborating with molecular geneticist Joshua Levin to test a handful of methods for sequencing RNA. The scientists were aiming to push the technologies to the brink of failure and see which performed the best. They processed samples with degraded RNA or vanishingly small amounts of the molecule. Eventually, Levin pointed out that they were sequencing less RNA than appears in a single cell.

To Regev, that sounded like an opportunity. The cell is the basic unit of life and she had long been looking for ways to explore how complex networks of genes operate in individual cells, how those networks can differ and, ultimately, how diverse cell populations work together. The answers to such questions would reveal, in essence, how complex organisms such as humans are built. “So, we’re like, ‘OK, time to give it a try’,” she says. Regev and Levin, who both work at the Broad Institute of MIT and Harvard in Cambridge, Massachusetts, sequenced the RNA of 18 seemingly identical immune cells from mouse bone marrow, and found that some produced starkly different patterns of gene expression from the rest1. They were acting like two different cell subtypes.

That made Regev want to push even further: to use single-cell sequencing to understand how many different cell types there are in the human body, where they reside and what they do. Her lab has gone from looking at 18 cells at a time to sequencing RNA from hundreds of thousands — and combining single-cell analyses with genome editing to see what happens when key regulatory genes are shut down.

The results are already widening the spectrum of known cell types — identifying, for example, two new forms of retinal neuron2 — and Regev is eager to find more. In late 2016, she helped to launch the International Human Cell Atlas, an ambitious effort to classify and map all of the estimated 37 trillion cells in the human body (see ’To build an atlas’). It is part of a growing interest in characterizing individual cells in many different ways, says Mathias Uhlén, a microbiologist at the Royal Institute of Technology in Stockholm: “I actually think it’s one of the most important life-science projects in history, probably more important than the human genome.”

Nature 547, 24–26 (06 July 2017) doi:10.1038/547024a

Expression of combinations of three different fluorescent proteins in a mouse brain produced ten different colored neurons. Individual neurons in a mouse brain appear in different colors in a fluorescence microscope. This “Brainbow” method enables many distinct cells within a brain circuit to be viewed at one time. 

Examples of experimental models of human cancer and their use. Modelling human cancer in other organisms allows us to observe and monitor the effect of therapies against the cancer before actually administering it to patients. This is best done in animal models as their biology is most similar to humans’. Non-animal, eukaryotic models are still useful for studying oncogenic processes in pathways that are conserved between the model and humans.

Genetic strategies

Classical transgenic mice use a tissue-specific promoter to drive the expression of an gene. The aim is to express the gene only in a particular tissue, and see if it causes tumours to form. It should also identify driver and passenger mutations. The downfall of this strategy is that oncogenes usually acts to de-differentiate the cell, so that would switch off the promoter that is driving its expression, resulting in a self-inhibitory feedback loop. Classical knockout mice have one or both copies of a gene knocked out in the germline, so all cells have the knockout. This is good for studying sporadic versus familial loss of heterozygosity events, but there is no control over where and when that gene is deactivated.

Inducible systems include the tet on and tet off systems, where tetracycline/doxycyline results in the induction (tet on) or inhibition (tet off) of the transcription of a gene. In tet on, doxycyline binds a transactivator that drives transcription; in its absence, there is no transcription. In tet off, the transactivator binds the locus without doxycycline; in its presence, the transactivator dissociates from the gene.

Cre-lox systems work by recombining DNA across lox sites. Any sequences occurring in between the lox sites are said to be “floxed”, and is removed upon Cre presence.

The ER-Tam system is a switchable system that works on the protein level. Proteins fused with the estrogen recetor (ER) are not functional unless tamoxifen is present. Tamoxifen can be removed to make the protein nonfunctional again.

in vivo models

Mouse models are widely used due to their evolutionary and genetic similarity to humans. Differences do exist, such as telomere length and mutation rate. They can be the recipient of xenografts and their cancers largely resemble human cancers.

Flies and yeast do not get cancer, but instead serve as models for oncogenic pathways. They can be subject to genetic and chemical screens to identify putative driver mutations causing pathway deregulation, and to identify drugs that can combat such mutations.

Culture

Cell cultures are generally not useful for monitoring tumours, because tumours are complex organs made of many cell types, and culture conditions usually maximise prolferation. Organoid culture allows growth of organs in 3D from a patient biopsy. This can be subjected to functional assays, drug screens, and genome editing for truly personalised therapy that is specific to the patient. However, this shares a common downfall with cell culture in that there is no contribution from the tumour microenvironment in a culture.

Further reading:

  • Sharpless, N.E.; DePinho, R.A. 2006. “The mighty mouse: genetically engineered mouse models in cancer drug development.” Nature Reviews Drug Discovery 5 (9):741-754.
  • Vidal, M.; Cagan, R.L. 2006. “Drosophila models for cancer research.” Current Opinion in Genetics & Development 16:10-16.
  • Xu, H.; Tomaszewski, J.M.; McKay, M.J. 2011. “Can corruption of chromosome cohesion create a conduit to cancer?” Nature Reviews Cancer 11 (3):199-210.

Mouse hippocampus

WHAT IS IT?
The hippocampus is found deep in the brains of many mammals, including humans. It’s named for its seahorse shape (in Greek, hippokampos literally means “horse sea monster”).

WHY IS IT IMPORTANT?
It helps us form memories and navigate space. It contains special cells called “place cells” that create a mental map of our environment. The hippocampus is also one of the first structures to suffer in patients with Alzheimer’s disease, which is characterized by memory loss. The number of patients with Alzheimer’s is predicted to triple by 2050.

WHERE DO WE GO FROM HERE?
Scientists at Harvard Medical School were recently able to re-create Alzheimer’s disease from human cells in a culture dish. This will “revolutionize drug discovery in terms of speed, costs and [disease relevance],” according to a senior co-author on the study.

Image by Chris Henstridge/MTA-KOKI/Nikon Small World.

High cocaine doses ‘can cause brain to eat itself’

A study of mice found that the drug can trigger out-of-control “autophagy”, a process by which cells digest themselves.

When it is properly regulated, autophagy provides a valuable cleanup service – getting rid of unwanted debris that is dissolved away by enzymes within cell “pockets”.

Dr Prasun Guha, from Johns Hopkins University School of Medicine in the US, who led the research published in the journal Proceedings of the National Academy of Sciences, said: “A cell is like a household that is constantly generating trash. Autophagy is the housekeeper that takes out the trash – it’s usually a good thing. But cocaine makes the housekeeper throw away really important things, like mitochondria, which produce energy for the cell.”

The scientists carried out postmortems that showed clear signs of autophagy-induced cell death in the brains of mice given high doses of cocaine. They also found evidence of autophagy in the brain cells of mice whose mothers received the drug while pregnant.

The scientists showed that an experimental drug called CGP3466B was able to protect mouse nerve cells from cocaine death due to autophagy. Since the drug has already been tested in clinical trials to treat Parkinson’s and motor neurone disease, it is known to be safe in humans. But much more research is needed to find out whether the drug can prevent the harmful effects of cocaine in people, said the team.

Co-author Dr Maged Harraz said: “Since cocaine works exclusively to modulate autophagy versus other cell death programs, there’s a better chance that we can develop new targeted therapeutics to suppress its toxicity.”

Image: There were clear signs of autophagy-induced cell death in the brains of mice given high doses of cocaine. Credit: OJO Images/Alamy

Source: Guardian (Press Association)

Peripheral nerves of a mouse embryo

Unlike the brain and spinal cord that are housed in protective bone, peripheral nerves connect regions of the body to the central nervous system like telephone cables. Peripheral nerves relay movement information from the brain to the muscles, for example, or sensory information from the skin to the brain. Remarkably, and also different from the brain and spinal cord, peripheral nerves have a tremendous capacity to regenerate when injured. Severed peripheral nerves grow about 1 mm per day (about an inch per month) until the two severed ends reconnect and innervate a once paralyzed muscle.

Image by Zhong Hua, Johns Hopkins University School of Medicine.

N6-methyladenine: A Newly Discovered Epigenetic Modification 

The majority of cellular functions are carried out by proteins encoded by specific genes present in cellular DNA. Genes are first transcribed to RNA which is then translated to proteins. The regulation of this process is important for maintaining correct cellular function. One of the ways that cells regulate gene expression is by epigenetic modifications to chromatin. The term “epigenetics” refers to reversible chemical modifications of DNA and histone proteins (DNA in the nucleus of eukaryotes is wrapped around histones) that affect the transcriptional status of genes. A number of histone modifications such as methylation and acetylation of lysine residues have already been discovered and characterized. Until recently; however, methylation of the 5 position of cytosine was the only known epigenetic DNA modification (A). Methylation of cytosine by DNA methyltransferases is associated with transcriptional silencing, while the removal of these methyl groups by TET enzymes is associated with transcriptional re-activation (B and C). In addition to controlling gene silencing, cytosine methylation also silences retrotransposons, a class of mobile genetic elements. If left unregulated, transposons can insert themselves into important regions of the genome and lead to mutagenesis.

Recently, N6-methyladenine, a new epigenetic modification, was discovered in mammalian cells. N6-mA had previously been discovered in prokaryotes and simple eukaryotes and was shown to function as a transcriptional activator. By contrast, a recent report published in Nature, has shown that N6-mA functions as a transcriptional silencer in mammalian cells, specifically in mouse embryonic stem cells. N6-mA primarily acts to silence the LINE-1 family of retrotransposons during early embryogenesis, which prevents genomic instability. The authors identified N6-mA by using a modified single molecule DNA sequencing technique. DNA bound to a specific modified histone protein was immunoprecipitated using an antibody against a specific histone modification (H2A.X), sequenced, and analyzed by mass spectrometry (D). This identified and determined the position of N6-mA. The authors then generated knockouts of the enzyme Alkbh1, which they believed may function as a demethylase for N6-mA. When Alkbh1 was absent from cells, they found an increase in the levels of N6-mA, showing that Alkbh1 functions as an N6-mA demethylase in vivo. This is important because epigenetic modifications are reversible. Genes can be turned off by methylation and then turned back on by removing the methyl group, so determining the enzyme responsible for the removal of N6-mA supports its role as an epigenetic modification.

For more information see:

http://www.nature.com/nature/journal/vaop/ncurrent/full/nature17640.html

As always, I’m happy to answer any questions or go into more detail.

06.18.17 - Sunday

My work in the lab is starting to pick-up in pace! I was feeling very overwhelmed these past few weeks because I’m so new to this area of research and I just had no idea what I was doing. I’m still not super confident in all the steps and procedures for everything but I at least understand why I’m doing certain things! 

The project I’m working on contains 3 components: a viral synthesis, general testing with a mouse cancer line cells, and the actual study on hippocampal neurons. It’s a lot but I’m excited about seeing it all come together!

-Cece 

Inner ear of a mouse

One of the most common genetic defects in human deafness is the disappearance of an important family of proteins: the claudins. Claudins are the most critical component of tight junctions (shown here in blue), the place where two adjacent cells meet. Imagine a tight circle of people linking arms to protect what’s inside; tight junctions are what protect a tissue from unwanted molecules or cells trying to pass through. When mice cannot make claudin, the tight junctions in the cochlea (the spiral-shaped portion of the inner ear) are disrupted, robbing them of their hearing sensitivity.

Image by Dr. Alexander Gow and Cherie Southwood, Wayne State University.

Watching thoughts — and addiction — form in the brain

More than a hundred years ago, Ivan Pavlov conducted what would become one of the most famous and influential psychology studies — he conditioned dogs to salivate at the ringing of a bell. Now, scientists are able to see in real time what happens in the brains of live animals during this classic experiment with a new technique. Ultimately, the approach could lead to a greater understanding of how we learn, and develop and break addictions. 

(Image caption: In a mouse brain, cell-based detectors called CNiFERs change their fluorescence when neurons release dopamine. Credit: Slesinger & Kleinfeld labs)

Scientists presented their work at the 252nd National Meeting & Exposition of the American Chemical Society (ACS).

The study presented is part of the event: “Kavli symposium on chemical neurotransmission: What are we thinking?” It includes a line-up of global research and thought leaders at the multi-disciplinary interfaces of the Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative with a focus on chemists’ contributions. The effort was launched in 2013 by the Obama Administration to enable researchers to study how brain cells interact to form circuits.

“We developed cell-based detectors called CNiFERs that can be implanted in a mouse brain and sense the release of specific neurotransmitters in real time,” says Paul A. Slesinger, Ph.D., who used this tool to revisit Pavlov’s experiment. Neurotransmitters are the chemicals that transmit messages from one neuron to another.

CNiFERs stands for “cell-based neurotransmitter fluorescent engineered reporters.” These detectors emit light that is readable with a two-photon microscope and are the first optical biosensors to distinguish between the nearly identical neurotransmitters dopamine and norepinephrine. These signaling molecules are associated respectively with pleasure and alertness.

Slesinger, of the Icahn School of Medicine at Mount Sinai in New York, collaborated on the project with David Kleinfeld, Ph.D., at the University of California at San Diego. Their team conditioned mice by playing a tone and then, after a short delay, rewarding them with sugar. After several days, the researchers could play the tone, and the mice would start licking in anticipation of the sugar.

“We were able to measure the timing of dopamine surges during the learning process,” Slesinger says. “That’s when we could see the dopamine signal was measured initially right after the reward. Then after days of training, we started to detect dopamine after the tone but before the reward was presented.”

Slesinger and colleagues will also share new results on the first biosensors that can detect a subset of neurotransmitters called neuropeptides. Ultimately, Slesinger says they’d like to use this sensing technique to directly measure these neuromodulators, which affect the rate of neuron firing, in real time.