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, includ

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 genes