Out-FOXOing High-Stage Neuroblastoma

Fluorescence microscopy of neuroblastoma cells.

In recent years, scientists have been hot on the trail of transcription factor FOXO3, tracing its involvement in various tumor-centric activities comprising the many trademarks of cancer, from drug resistance to metastasis to tumor angiogenesis.

FOXO3 is a member of the O sub-class of the forkhead box family of transcription factors. The forkhead box (FOX) family is characterized by a fork head DNA-binding domain (DBD), comprised of around 100 amino acids. They have also proven themselves to be a family of many hats, functioning in diverse roles ranging from metabolism, immunology, cell-cycle control, development, as well as cancer (1). The forkhead box O (FOXO) sub-class alone has demonstrated involvement in a variety of cellular outcomes, from drug resistance and longevity to apoptosis induction.

Due to its pro-apoptotic and anti-proliferative proclivity, FOXO3 has been previously identified as a tumor suppressor gene. However, more and more studies have begun to flip the narrative on FOXO3, portraying it more as a devoted henchman, due to its roles in drug and radiotherapy resistance, cell-cycle arrest and long-term maintenance of leukemia-initiating stem cells in a variety of cancer types, including breast cancer, pancreatic cancer, glioblastoma, and both acute and chronic myeloid leukemia.

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Targeted Gene Modification in Prairie Voles Using CRISPR and pGEMĀ®-T Easy Vectors

As the number of children diagnosed with autism spectrum disorder (ASD) continues to rise, the search for a cause continues. Scientists have been studying genetically modified oxytocin receptors, which have shown promise as a target for studying ASD-related behaviors. One of the obstacles to designing robust scientific experiments for investigating potential ASD causes or treatments is the lack of a truly appropriate model organism for social behaviors in humans (1). Sure, there are the traditional lab rats and lab mice that demonstrate a certain level of social behaviors. However, there has been a loss of natural social behaviors in common lab mice strains because of the reduction in genetic complexity from inbreeding and adaptation to captivity (2). These animals cannot fully represent the depth of human social behaviors, including the ability of humans to form lasting social bonds (1).

Enter: The prairie vole (Microtus ochrogaster).

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Reliable DNA Purification from 3D Cell Cultures

Traditionally, scientists have relied on flat, two-dimensional cell cultures grown on substrates such as tissue culture polystyrene (TCPS) to study cellular physiology. These models are simple and cost-effective to culture and process. Within the last decade, however, three-dimensional (3D) cell cultures have become increasingly popular because they are more physiologically relevant and better represent in vivo conditions.

A spheroid of ~1,000 human liver cells. Image provided by Insphero.
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Choosing a Tag for Your Protein

You have identified and cloned your protein of interest, but you want to explore its function. A protein fusion tag might help with your investigation. However, choosing a tag for your protein depends on what experiments you are planning. Do you want to purify the protein? Would you like to identify interacting proteins by performing pull-down assays? Are you interested in examining the endogenous biology of the protein? Here we cover the advantages and disadvantages of some protein tags to help you select the one that best suits your needs.

Immunofluorescent detection of HiBiT-tagged proteins in CRISPR-edited cell pools and clones using the Anti-HiBiT Monoclonal Antibody.
CRISPR-Cas9 editing knocked-in HiBiT at the endogenous locus of proteins with varying subcellular localization. Fixed CRISPR-modified clones or pools of cells were imaged by immunofluorescent staining using the Anti-HiBiT Monoclonal Antibody (red) and Hoechst dye (blue). Panel A. VCL-HiBiT pool. Panel B. SMARCA4-HiBiT clone. Panel C. HDAC2-HiBiT clone. Panel D. HSP90B1-HiBiT pool.

Affinity Tags

The most commonly used protein tags fall under the category of affinity tags. This means that the tag binds to another molecule or metal ion, making it easy to purify or pull down your protein of interest. In all cases, the tag will be fused to your protein of interest at either the amino (N) or carboxy (C) terminus by cloning into an expression vector. This protein fusion can then be expressed in cells or cell-free systems, depending on the promoter the vector contains.

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To Sleep, Perchance to Clean

While you and I are getting some shut eye each night, things are happening in our brains. Good things. Therapeutic things.

Think of it as brainwashing of a sort. There is a multiplicity of brain activities going on during sleep, and a November 1 paper in Science shows for the first time when and where in the brain these activities occur, and how they are connected.

CSF washes through the brain.

Here’s a bit of backstory.

To assess both the progression and pathogenesis of Alzheimerā€™s disease (AD), as well as the efficacy of AD drugs in clinical trials, there has been interest in the concentrations of amyloid-beta (AĪ²) and tau protein in cerebral spinal fluid (CSF).

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T-Vector Cloning: Questions, Answers and Tips

Blue/White colony screening helps you pick only the colonies that have your insert.

Q: Can PCR products generated with GoTaq DNA Polymerase be used to for T- vector cloning?

A: Yes. GoTaqĀ® DNA Polymerase is a robust formulation of unmodified Taq Polymerase. GoTaqĀ® DNA Polymerase lacks 3ā€™ ā†’5ā€™ exonuclease activity and displays terminal transferase activity that adds a 3ā€² deoxyadenosine (dA) to product ends. As a result, PCR products amplified using GoTaqĀ® DNA Polymerases (including the GoTaqĀ® Flexi and GoTaqĀ® G2 polymerases) will contain A-overhangs which makes them suitable for T-vector cloning with the pGEMĀ®-T (Cat.# A3600), pGEMĀ®-T Easy (Cat.# A1360) and pTARGET™ (Cat.# A1410) Vectors.

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MSI Testing of Tumor Cells for Better Tailored Treatment

3D artistic rendering of mismatch repair. Microsatellite instability (MSI)  which can result from defects in mismatch repair is a biomarker for some cancers

There are as many different cancers as there are people with cancer. Unlike infectious diseases, which are caused by pathogens that are foreign to our bodies (bacteria, viruses, parasites), cancer cells arise from our bodyā€”our own cells gone rogue. Because cancer is a dysfunction of a personā€™s normal cells, every cancer reflects the genetic differences that mark us as individuals. Add to that environmental influences like diet, tobacco use, the microbiome and even occupation, and the likelihood of finding a ā€œsingleā€ pharmaceutical cure for cancer becomes virtually impossible.

But, while looking for a single cure for all cancers may not be a fruitful activity, defining a best practice for understanding the genetic and protein biomarkers of individual tumors is proving worthwhile.

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The Future of Synthetic Biology: A Recap of iGEM 2019

After attending the iGEM Giant Jamboree last year and being completely blown away by the projects presented (check out this article or this one), I didnā€™t think Iā€™d be as astonished this year. I attributed part of the awe I felt over the caliber and quality of the projects to my wide-eyed naivetĆ©, having never attended the event before. The second time around, the ā€œfirst-timeā€ novelty long worn off, I didnā€™t expect to feel that same level of amazement.

I couldnā€™t have been more wrong.

After three days of impressive presentations, I once again felt that same astonishment as I prepared to watch the presentations of the 6 finalists. With good reasonā€”the projects presented by the six finalists completely blew my mind!

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Tales from the Trenches: Career Growth in Biotechnology

Building a successful career in the biotechnology industry is really just a series of transitions from one role to another. But the devil is in the detailsā€”when to make a change, how to create opportunities and who can be your champion as you pivot. So how do you navigate these factors to keep your career goals on course?

Bob Weiland answers a question posed by Michele Smith at the MS Biotech Alumni Symposium.

I recently attended a symposium (presented by the University of Wisconsin Master of Science in Biotechnology Program, of which Iā€™m an alum) that addressed this topic through the lens of one individual with a storied career in the industry. Bob Weiland currently serves on the Board of Directors for CymaBay Therapeutics. He has held various roles, from sales and marketing to operations and strategy, within large, established companies (Abbot, Baxter, Takeda) and smaller ones (Pacira Pharmacueticals). He drew on this wide-ranging experience to provide advice to professionals at all career stages.

Bob began the talk by declaring that there will be points in your career when you reach a ā€œhard spotā€ and will need to transition, whether to a new role, company or even industry, to meet your career goals. He suggested a good starting point is simply to be thinking about making a change. But in the same breath he emphasized, ā€œWhat are you doing about it?ā€ He identified four distinct actions that you can take to ensure role changes and career transitions support your professional growth and development.

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How should I evaluate DNA isolated from FFPE samples to ensure success?

Part two of three. You can read part 1 here.

Formalin-Fixed Paraffin embedded (FFPE) samples are being used in increasing numbers of molecular assays. In my last blog I discussed some of the pre-analytical variables that can affect results obtained when using FFPE samples. Laboratories can increase the quality of downstream results by controlling variables where possible. While exacting control over the sample acquisition and fixation process can improve results, quality testing of incoming samples is a crucial step in assuring optimal results. There are numerous methods that can be used to evaluate the quality of samples and they can provide different information that can be used to assess sample integrity and suitability for different applications.

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