Cyagen Biosciences

The nervous system presents several unique challenges that make it a difficult system to study experimentally. At a structural level, the brain has a complexity that is orders of magnitude greater than other organs, and even the peripheral nervous system is profoundly complex. At a cellular level, neurons and accessory cells have extreme morphologies, physiological properties, and sensitivities that make them challenging to manipulate experimentally. In addition to these difficulties, the brain is also protected by the so-called blood-brain-barrier (BBB). The endothelial cells forming the vessels of the brain are highly selective in regulating passage into the cerebrospinal fluid, preventing the entry of viruses and bacteria, while regulating the transport of hormones, ions, drugs, and other molecules. The BBB hampers the effectiveness of many in vivo experimental techniques. For example, most drugs and viral vectors delivered into the blood cannot effectively cross the BBB and infi...

Macrophages are the first line of defense against infections, playing important roles in consuming pathogens and in regulating inflammation. In general, these functions help maintain a healthy organism, keeping infections at bay and promoting healing. However, it is now well known that inflammation is associated with cancer progression, and that the presence of macrophages within a tumor often correlates with poor prognosis (1). But, the relationship between inflammation and cancer progression is poorly understood, and much is still unknown about how macrophages may contribute to tumor growth or metastasis. Using in vivo animal models of melanoma invasion, a new study has uncovered a surprising way that macrophages may be affecting cancer cells. Using zebrafish and mouse models, high-resolution imaging, and an elegant Cre/Lox fluorescent reporter strategy, Roh-Johnson et al. discovered that macrophages actually exchange cytoplasm with melanoma cells in live animals (2). They observed...

Either shRNA-mediated knockdown or nuclease-mediated knockout (e.g. CRISPR or TALEN) can be valuable experimental approach to study the loss-of-function effects of a gene of interest in cell culture. In order to decide which method is optimal for your specific application, there are a few things you should consider. Mechanisms Knockdown vectors: Knockdown vectors express short hairpin RNAs (shRNAs) that repress the function of target mRNAs within the cell by inducing their cleavage and repressing their translation. Therefore, shRNA knockdown vectors are not associated with any DNA level sequence change of the gene of interest. Knockout vectors: CRISPR and TALEN both function by directing nucleases to cut specific target sites in the genome. These cuts are then inefficiently repaired by the cellular machinery, resulting in permanent mutations, such as small insertions or deletions, at the sites of repair. A subset of these mutations will result in loss of function of the gene...

Direct transfection of cells with the viral vector (rather than using live virus) may facilitate expression of your gene of interest (GOI), but there are a number of complications (see below). We therefore recommend that viral vectors be used for production of live virus, and not for direct transfection of cells. Virus transduction can usually deliver DNA into target cells more efficiently than plasmid transfection. When using retrovirus such as lentivirus or MMLV, the viral genome can integrate into the host cell genome so that genes carried on virus can be stably expressed. By contrast, transfected vector plasmids only have transient expression in the cells since they do not integrate into the host genome. For retroviral vectors, comparing to virus transduction that has low copy number in the host genome, direct transfection of plasmids can often result in very high copy number in cells, which leads to very high expression levels of the genes carried on the vector. However, this can ...

Introduction: Although multicellular organisms are made up of many cell types, all with essentially identical genomes, cells rarely interconvert between cell types. Once a cell acquires a specific cell fate, it generally does not assume the phenotype of an alternate cell type. However, in certain developmental and repair processes, cells do undergo reprogramming to alternate cell identities, demonstrating that cell type is somewhat plastic (1).  There is now a concerted effort by many labs to study and manipulate cell type plasticity, and a lot of progress has been made (2). Recently, there have been some major breakthroughs in reprogramming, allowing clinically relevant cell types to be generated in animal models in vivo. Here are a couple recent examples, both utilizing viral vectors to trigger reprogramming. Replacing lost liver cells In most types of chronic liver disease, the accumulation of fibrosis and associated hepatocyte loss can lead to serious health issues, in...

For CRISPR-mediated genome editing, Cas9 nuclease is directed to the target site of site-specific guide RNA (gRNA) in the genome to create DNA cleavage. In most cases, to generate simple gene knockout, a single gRNA can be used together with Cas9 to generate a double-strand break (DSB), which is then inefficiently repaired by the non-homologous end joining (NHEJ), resulting in permanent mutations, such as small insertions or deletions, at the site of repair. A subset of these mutations will result in loss of function of the gene of interest due to frame-shifts, premature stop codons, etc. Dual gRNAs can be used if Cas9_D10A nickase is being used to target the two opposite strands of a single target site. In this approach, the nickase enzyme will generate single strand cuts on both strands, one guided by each of the two gRNAs, resulting in DSBs at the target site. Generally, this method reduces off-target effects of CRISPR/Cas9 expression because targeting by both gRNAs is necessary fo...

Common viral vectors used in biomedical research include lentivirus, Moloney murine leukemia virus (MMLV), adenovirus, and adeno-associated virus (AAV), each with its advantages and disadvantages. Many factors affect the decision on what type of viral vector to use in your experiment. The key considerations include: Does the virus have the tropism for the target cells (namely, can it efficiently infect target cells)? Are the cells dividing or non-dividing? Do you want transient transduction or stable integration into the host genome? What transduction efficiency is needed? Do you need to use a customized promoter to drive the gene of interest? Will your vector be used in cell culture or in vivo? Will an immune response to the virus affect your experiment?  Lentivirus Lentivirus is a type of retrovirus. Upon infecting cells, the RNA genome of the virus is reversely transcribed and then permanently integrated into the host genome, thus allowing long-term stable expression of ge...

Introduction: Both CRISPR and TALEN systems have been harnessed to edit genomes of cultured cells and model organisms. Both systems can be used to knock out genes, or to knock in point mutations or insertions, but these two systems are different in several ways and have their own pros and cons. Mechanisms CRISPR: The CRISPR system uses a site-specific guide RNA (gRNA) to direct the Cas9 nuclease to its target site in the genome to create DNA cleavage. The target sequence is typically ~20 bp long, and sites containing a few mismatches may still be recognized and cleaved. TALEN: The TALEN system employs a pair of chimeric proteins, each composed of a TAL effector DNA-binding domain (recognizing a specific sequence) fused to a FokI nuclease domain. The pair of proteins are designed to bind to a pair of target sites in the genome, each ~18 bp long and flanking a 14-20 bp spacer. Upon binding to DNA, the Fokl nuclease domains on the pair of proteins are able to dimerize, which in tu...

Introduction: For decades, new innovations in stem cell biology and genetic manipulation have driven a wave of proposed treatments based on these technologies. Although there has been intense speculation and expectation about when and how these methods will finally begin to show true potential for impacting patients' lives, there is still only one gene therapy which has been approved by the US FDA. However, the results of a recent clinical trial suggest a new type of gene therapy may soon be available. In a very promising new study, doctors have applied both gene and stem cell therapies together to stop a childhood neurodegenerative disease known as cerebral adrenoleukodystrophy (ALD). This is a severely debilitating disease affecting nerve myelination, and patients diagnosed with ALD usually do not live more than a decade after the disease is identified. Until now, the only viable treatment for children afflicted with this genetic disorder has been a hematopoietic stem cell (H...

Introduction: Alcoholism is a complex disease affecting hundreds of millions of people worldwide (1). Despite having devastating consequences on physical and mental health, and indirectly impacting families and society, treatment options are limited and often ineffective (2). Current evidence suggests that environmental and genetic factors both equally influence an individual’s susceptibility to alcoholism (3). Genetics of alcohol metabolism Interestingly, people with certain genotypes are significantly less likely to become alcoholic, likely due to unpleasant, aversive effects they experience when consuming alcohol (4-7). The most well-understood examples are variants of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) genes. These enzymes are sequentially involved in metabolizing ethanol to acetaldehyde and then acetate. Individuals who carry mutations in these ALDH genes can accumulate acetaldehyde in the blood after ethanol consumption producing strong effects, inc...

Introduction:  Ever since the cloning of channelrhodopsin-2 (ChR2), there has been an explosion of interest in applying optogenetic methods to all aspects of neuroscience (1). Initially, ChR2 was used to control neuronal activity in vitro (2,3), and shortly thereafter in animal models (4). More recently, light-activated systems have even made their way to human clinical trials. Scientists and clinicians continue to develop and brainstorm new uses for this powerful set of tools. Here are a few of the interesting applications of optogenetics that may soon directly impact human health, and in some cases already have. Restoring sight to the blind Probably the first proposed use of optogenetics to treat a disease was by Zhuo-Hua Pan, who hypothesized that ectopic expression of light-gated channels in patients with retinal degeneration could restore light-sensitivity to the eye. This approach proved successful in mouse models when adeno-associated virus (AAV) was used to express ChR2...

During the past several years, there has been an increasing public awareness of mental illness and their societal and health consequences. Eating disorders represent one of the most damaging groups of mental illnesses. Current estimates are that anorexia nervosa claims the life of an astonishing 10-20% of patients, and increases mortality rate by 6-fold. Based on these figures, anorexia is the single most deadly mental illness (1). Another surprising fact about eating disorders is that risk of anorexia and bulimia appears to be about 50% attributable to genetics (2). Despite this, there has been little progress toward identifying specific biological factors which are causal for eating disorders. There is good news, however, as some recent studies using animal models are beginning to provide much-needed insight into these complex and tragic illnesses. Researchers from Columbia University have reported a mouse model that recapitulates many aspects of human anorexia nervosa, includi...

Around 20 million years ago, for unknown reasons, wild pigs lost an important metabolic gene known as uncoupling protein 1 (UCP1). In most mammals this gene plays a major role in generating body heat for thermoregulation, and lack of UCP1 has led to some unusual physiological traits in all modern domestic and wild pig species (1). Unlike most mammals, pigs generate heat in response to cold temperatures primarily by shivering, instead of by increasing metabolic rate. In fact, pigs seem to entirely lack brown adipose tissue, the type of fat specialized in thermoregulation. As a consequence, piglets are exceptionally susceptible to cold-related death, and adult pigs tend to accumulate excess fat as a result of their unusual metabolic traits. From an agricultural perspective, there are significant economic costs due to neonatal mortality, heating animal barns, and feed costs associated with excess fat production. Piglet death also represents an obvious animal welfare issue. A team of s...

Around 100 million people worldwide have red hair due to the variants of the melanocortin-1 receptor (MC1R) gene they carry. In addition to affecting hair color and skin pigmentation, red-hair-color (RHC) MC1R variants also significantly increase sun sensitivity and the risk of skin cancers such as melanoma. Until now we have known almost nothing about how MC1R is modulated by UV radiation, or how certain MC1R alleles increase the risk of skin cancer. But, in a recent paper, researchers seem to have finally found the elusive answer, and potentially also discovered new ways to prevent sun-induced melanoma. Chen et al. found that MC1R undergoes a lipid modification called palmitoylation, and that this modification controls the development of melanoma. They used an impressive combination of mouse models, cell culture, and biochemical methods to elucidate a multistep pathway in which activation of ATR kinase by UV radiation leads to phosphorylation of the protein-acyl transferase ZDHHC13...

Last month, the Nobel Foundation recognized Jeffrey Hall, Michael Rosbash, and Michael Young "for their discoveries of molecular mechanisms controlling the circadian rhythm" (1). This 24-hour "clock" influences many physiological processes, and has a well-understood biochemical basis elucidated by the work of many researchers over the past few decades. Interestingly, in addition daily circadian cycles, many organisms also display physiological cycles repeating twice a day. Most obviously, coastal animals possess a powerful "circatidal clock", which oscillated with the 12.4-hour ebb and flow of the tides, influencing locomotion, metabolism, and many other physiological processes (2). Even in humans, body temperature, hormone levels, blood pressure, and other functions fluctuate with a predictable 12-hour period, and some human diseases have even been associated with perturbed 12-hour cycles (3-9). In a fascinating recent study, researchers used mathem...

Biology research relies on the use of model systems. The type of system used can affect the scope and limitations of a research project, and also affects the cost, timeframe, and interpretation of experimental results. There is ongoing debate in many fields as to whether animal models or cultured cells are superior model systems. Of course, the truth is that both are necessary, with advantages and disadvantages, and for some research goals, only one type of model system will do the job. Here we will discuss a few of the issues raised in the debate between cells and animal models. Scaling: Cultured cell experiments are highly scalable; more experiments can be done more rapidly than using animals. For example, potential cancer drugs are typically tested on large panels of hundreds of different cell lines with known mutations1. This would not be feasible with animals, but animal models allow testing on intact physiological systems of many cell types simultaneously. For example, mouse...

Blindness is a major public health problem. Nearly 40 million people worldwide are functionally blind, while over 250 million people suffer from visual impairment. The major causes of both acquired and hereditary blindness in the developed world are age-related macular degeneration and retinitis pigmentosa, which both share a common pathophysiology. In these conditions, the photoreceptors of the outer retina degenerate irreversibly, leaving the inner retina intact but unable to process visual input. Due to the anatomical and physiological similarities between visual systems across mammalian species, rodent models are incredibly powerful tools for the testing and development of therapies to treat blindness. Here are a few examples of how mouse models are being used to give sight to the blind. AAV-based gene therapy was used by Cehajic-Kapetanovic and colleagues to introduce a human rhodopsin gene into the cells of the inner retina in a mouse model of retinitis pigmentosa (hereditary ...

Tobacco use represents a major worldwide health problem. Around one fifth of the world's population regularly smokes tobacco and nearly half of these people will die from tobacco-related illnesses (1). And yet, nicotine addiction is extremely difficult to overcome. A study just published by Xue et al., from The Scripps Research Institute, describes a new biochemical tool that could eliminate the addictive power of nicotine, and make quitting much easier (2). This team of biologists and chemists made use of bacteria known as Pseudomonas putida, isolated from tobacco fields (3). These bacteria consume nicotine as a food source, and an enzyme known as NicA2 is a primary component of their nicotine metabolism. Using a gene from P. putida, Xue et al. were able to purify recombinant NicA2 enzyme capable of degrading nicotine. They anticipate that the NicA2 enzyme may soon be used as a treatment for nicotine addiction. The hope is that NicA2, if administered to smokers, could dest...

Introduction : Animal models play a vital role in modern biomedical research. In particular, mouse and rats are profoundly important. However, mice and rats are imperfect models of human diseases and are poor platforms on which to test potential human therapeutics. In fact, standard rodent models become more limited as the research questions become more clinically relevant.These shortcomings are frequently due to sequence differences between orthologous genes that play important roles in certain biological processes underlying human health issues, such as cancer, diabetes, and mental health, and also due to a lack of cross-reactivity between therapeutics and the mouse orthologs of their human targets. Humanized rodents Humanization of mouse and rat alleles is now a powerful approach to overcome these limitations and make mice and rats which are better suited for human biomedical research. A humanized allele consists of a rodent gene which is eliminated and replaced by the corre...

Introduction: For more than a century, rodents have played a pivotal role in biomedical research, and provided experimental systems for investigating mammalian biology. Although rats were once the most widely used organism in medical research, mouse research has come to the forefront of scientific and media attention due to the power of mouse genetics and the availability of mouse embryonic stem cells (ESCs). However, with a fully sequenced rat genome and new genome engineering technologies such as TALENs and CRISPR/Cas9 , rats are now nearly as genetically tractable as their smaller cousins. Advantages of rats Rats are vastly superior to mice as a model system in many avenues of research. Studies in toxicology, hypertension, nutrition, pharmacology, behavior, cancer, osteoporosis, and many other fields strongly benefit from the larger size, superior cognition, and more human-like physiology of rats. Here are some specific examples: The ovariectomized rat is the most commonly...

Over the past several years, huge advances in genome editing technologies have fueled an almost palpable excitement about the future of genome engineering. Beginning with the discovery of zinc-finger nucleases, and followed recently by the description of TALEN and CRISPR genome-editing systems, the possibility of literally rewriting a genome has quickly gone from dream to reality. An incredibly exciting potential use of genome-editing technologies is to correct genetic mutations that cause diseases. Everyone aware of CRISPR and TALEN technology has obviously considered this possibility, and the medical and biotechnology fields are working towards the development of the first genome editing-based treatments. Now, for the first time, researchers have demonstrated that CRISPR can be used to eliminate a disease-causing mutation from cells in an animal model of a human disease. These three studies, published in the most recent issue of Science, show that the CRISPR system can ef...

In solving the mystery of gene function, there is no more important clue than the phenotype of inactivating the gene of interest. With a plethora of methods available, researchers must first determine what approach is best for their specific scientific questions and experimental systems. For over a decade, RNA-interference-based methods of gene knockdown (i.e. RNAi & shRNA) have provided a wealth of insight into gene function, but in recent years the advent of CRISPR- and TALEN-based methods now allow genome editing to be used to quickly and efficiently test the effect of gene knockouts. Here, we review the advantages and disadvantages of these approaches, and describe some experimental situations in which one approach is better than another, focusing primarily on CRISPR/Cas9 and shRNA. For a discussion of the relative advantages of CRISPR/Cas9 versus TALEN , see our prior Newsletter on this topic. CRISPR Knockout In this method, a guide RNA (gRNA) homologous to an 18-22nt ta...

The gold standard for genetically engineering mouse models is ES-cell based homologous recombination. However, this approach is very time-consuming and costly. Recently, TALEN and CRISPR/Cas9 systems have been harnessed to edit genomes of cultured cells, mice and rats1,2. Both systems can be used to create knockouts, and to introduce point mutations or small insertions, but each has distinct advantages (see Table 1). TALENs are chimeric proteins composed of site-specific DNA-binding domains fused to the non-specific endonuclease FokI. CRISPR/Cas9 uses a site-specific single guide RNA (sgRNA) to direct the Cas9 nuclease to its target locus. TALEN CRISPR/Cas9 Origin Plant pathogenic bacteria (Xanthomonas) Diverse bacteria Components Pairs of TALE-FokI fusion proteins Guide RNA and Cas9 Efficiency High High but variable Off-target effects Minor Moderate to high Target site availability No restriction Requires PAM (NGG) motif Time required for vector engineering One week 1-3 day...