Cyagen Biosciences

Loss-of-function screening using shRNA libraries is a powerful way to identify genes involved in almost any biological process. Over the past decade, shRNA screens, both in vivo and in cell culture systems, have generated many important discoveries. Knockdown screening has become a go-to ‘workhorse’ method for lead identification and gene network studies across many fields of biology. Whole-genome knockdown libraries are frequently used, as well as smaller libraries targeting subsets of genes, such as specific pathways or key biological regulators. Here are a few examples of major shRNA screening discoveries from the past few years: Peroxisomes have a role in cholesterol transport It has long been known that cholesterol undergoes intracellular transport, but the mechanism underlying this process remained unknown. Chu et al. developed an amphotericin B-based assay for impaired LDL-transport, and used it in a genome-wide, lentivirus-based shRNA screen (1). They found that knockdown of

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

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

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

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

Viruses are pseudo-living organisms which inhabit and influence virtually every type of plant and animal cell. In some contexts, viral infection has obvious and profound consequences for the host organism, such as causing disease. However, many viral infections go completely unnoticed, with infections having essentially no phenotype or having an effect only long after the initial infection. Several examples of chronic, unnoticed viral infections affecting human health have been discovered. In several cases these long-term effects have been implicated in major, worldwide health problems. Cytomegalovirus (CMV) is one of the most common human virus, being present in around 75% of adults worldwide. Once acquired, CMV infection persists lifelong in a dormant state. CMV infection is a known risk factor for high blood pressure and has been implicated in atherosclerosis, and people who test positive for human CMV infection are at increased risk of cardiovascular diseases and heart attack