...develop therapies to treat disease in the lung. Chapter 2: Balancing research with medical practice as an MD...engineered cells in therapy. The lungs are difficult, but other tissues like the retina have cell-based therapies...Chapter 9: Using stem cells as in-vitro models of disease. How Darrell is using stem cells from patients to...sharing reagents, cell lines, data, and knowledge without exclusivity. Chapter 11: The Stem Cell Bank (CReM ...the lungs, and, among other projects, using stem cells to develop in vitro models for lung disease. Darrell...what drives him as a praticing physician and stem cell researcher. Darrell's ultimate goal - to develop...means to treat disease through tissue repair, stem cells, gene therapy, and tissue engineering. How the multidisciplinary...

...), 1668. https://doi.org/10.1038/s41467-019-09656-2 More resources on the Addgene blog CRISPR101: Validating...hematopoietic stem cells with a lentiviral vector containing a gRNA. These transduced stem cells can be implanted...Annotation of genes in immune cells typically involves the creation of germline knockout mice, which ...CRISPR-based systems enable gene knockout in immune cells in a high-throughput manner, but these systems have..., ubiquitous knockout of single genes in immune cells in vivo (LaFleur et al., 2019a and LaFleur et al...many genes have different functions in different cell types, at different times, and in combination with... L-CHIME Knocks out a single gene in defined cell types (lineage-based) pXPR_070 Violet-excited...

...SARS-CoV-2 spike pseudotyping, visit the SARS-CoV-2 Pseudotyped Virus collection page. SARS-CoV-2 detection...of SARS-CoV-2 entry and become upregulated during viral infection.  Expression of SARS-CoV-2 viral proteins...pseudotyping The SARS-CoV-2 virus depends on its spike protein to bind ACE2, the host cellular receptor. To more...point for SARS-CoV-2 researchers to investigate how the virus interacts with human cells. Addgene’s mammalian...genes are important for SARS-CoV-2 entry into human lung epithelial cells (Biering et al., 2021). In their...Wesemann DR (2021) Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike bioRxiv. https://...labs in over 75 countries. Figure 1: SARS-CoV-2 proteins and other components. Image from Maya Kostman...

...(I), or sparse (S) labeling of the cell population. Figure 2: Dense (c), intermediate (d), and sparse...single genetically-defined subset of cells. By labeling fewer cells in a population, it’s easier to visualize...non-overlapping cells. While transgenic animals are commonly used to specifically manipulate a cell type of interest...expressed. In cells where the stop cassette remains, the effector is not expressed and the cells are not labeled...sparse labeling of the cell population that expresses SPARC and the fraction of cells that will be labeled... interest in an organism, all cells of that type are affected. Previous tools to overcome this restraint... of the gene construct. A predictable number of cells will express the effector or retain the stop cassette...

...Everyone. Case study 1: Cell lines Many of the reagents used to investigate SARS-CoV-2 were developed for ...different organisms to SARS-CoV-2 (Chu et al., 2020). An additional cell line used in this study came from...tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for...fast access to the tools required to study SARS-CoV-2, but with many academic research labs closed during...biobanks, are crucial for the advancement of SARS-CoV-2 research because they provide rapid access to validated...that science is truly a global endeavor. Case study 2: Plasmids Another example of reagents that have broad...applications but have proven useful to the study of SARS-CoV-2 are the lentiviral production and packaging vectors...

....9 (parental) 46 +/- 0.7% AAV.PHP.S 82 +/- 2% Table 2: Transduction efficiency of AAV.9 versus AAV.PHP.S...figure 2) to allow for efficient IV delivery of virus to the CNS or PNS (figure32, tables 1 & 2). These...that cells are labeled with their own unique color; and 2) low levels of labeling so that a cell’s projections...in tables 1 and 2, with a cell-type specific promoter or enhancer now allows for cell-type-restricted ... overall (GFP+ cells that also stained for a neuronal marker). Check out tables 1 and 2 below for a summary...high dose to maximize the number of cells that are labeled. Part 2: An AAV expressing the inducer (i.e...require the generation of a transgenic mouse. 2. Cell-type-specific restricted transgene expression in...

...pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 N protein  pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro...SARS-CoV-2 M protein pLVX-EF1alpha-SARS-CoV-2-orf3a-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 ORF3a... SARS-CoV-2 protein expression The Krogan lab quickly shared plasmids expressing SARS-CoV-2 proteins at...pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro) and NSP2 (pLVX-EF1alpha-SARS-CoV-2-nsp2-2xStrep-IRES-Puro...months: Lentiviral preps for expressing SARS-CoV-2 proteins ChR2 for targeted expression DREADDs GFP ...Serotype Name 83899 AAVrg pAAV-mDlx-GCaMP6f-Fishell-2  Additional resources on the Addgene blog Read... results, but also confirm expression in target cells. While the various AAV serotypes can be used to ...

...the image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy attempts to solve two ...Fluorescence microscopy. Nature methods 2, 910-919 (2005). PubMed PMID: 16299476. 2. Conchello JA, Lichtman JW. Optical...provide a 2-10 fold increase in resolution, down to 10s of nanometers. Thick dynamic samples Ex: 3D cell cultures...enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy...photobleaching and a reduction in signal intensity. 2-Photon microscopes restrict excitation (and bleaching...can be seen exiting through the tissue. Therefore, 2-photon allows for deeper imaging into thick tissues...imaging objective that collects fluorescence. Like 2-photon microscopy, only a single focal plane of the...

...Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct epitopes on the SARS-CoV-2 nucelocapsid protein...repository of tools for SAR-CoV-2 research. Two new antibodies, Anti-SARS-CoV-2 Nucleocapsid Protein [mBG17...Anti-SARS-CoV-2 Antibodies here! Terry, J.S., et al. (2021). Development of a SARS-CoV-2 nucleocapsid ...domain, PE7 (Figure 2).  PE7 shows enhanced editing efficiencies in a number of cell types at different...Figure 3).      Figure 3: HeLa cells transduced with SARS-CoV-2 nucleocapsid protein (Plasmid #141391...prime editor PE7 Antibodies to facilitate SARS-CoV-2 research New viral vector preps of Voltron2 voltage...editor. Find PE7 plasmids here!     Figure 2: PE7 improves prime editing. A) Schematic of PE7 prime...

...SARS-CoV-2 infects host cells through binding the human angiotensin converting enzyme 2 (ACE2) cell-surface...severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus may acquire mutations to escape ...Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and sequencing. From Starr et al Cell., 2020. (B) Mapping...designed so that the SARS-CoV-2 spike RBD is displayed on the yeast cell’s surface. The Bloom lab used ...Pooled library to study antibody escape in SARS-CoV-2 Recombinant antibodies for aminoacyl tRNA synthetases...viral preps Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz As multiple... binding ACE2 is the target of most anti-SARS-CoV-2 antibodies. In the library, each plasmid encodes the...

...Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. Cell 184:2372-2383...months, we’ve seen the emergence of several SARS-CoV-2 variants coincide with increased vaccination efforts...They were able to tweak this assay to study SARS-CoV-2 spike variants. “We created a series of spike expressing...relative ability to neutralize.”   Figure 2: Sera used in the study came from individuals who ...This sera was used in the high-throughput SARS-CoV-2 pseudovirus neutralization assay. Neutralization was...viruses are quite genetically distinct from SARS-CoV-2 and its variants. Yet, the B.1.351 variant, which ...effectiveness of current vaccines against SARS-CoV-2 variants, this study was the first to compare so many...

...Illustration of SARS-CoV-2 from Philippa Steinberg for the IGI.   What SARS-CoV-2 plasmids from Ginkgo ...share over 1,850 SARS-CoV-2 plasmids to help academic and industrial labs accelerate research and to help ...request? Currently, plasmids containing all SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...bacterial expression systems, Ginkgo synthesized SARS-CoV-2 expression plasmids in a high copy backbone with a...high level expression and purification of SARS-CoV-2 proteins for downstream experiments and offer flexibility...optimized, full length and truncated versions of SARS-CoV-2 S (spike protein) cloned into a CMV driven expression...first deposited set of plasmids containing SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...

...comprehensive SARS-CoV-2 virus-human interaction network in which we expressed 26 of the 29 SARS-CoV-2 viral proteins...understanding how SARS-CoV-2 interacts with human proteins. For example, we used HEK293T cells in our first study...all became acutely aware of the threat of SARS-CoV-2. Like many in the science community, we’ve been motivated...identifying these potential human helpers of SARS-CoV-2 (especially the ones that are druggable) is a key ... joining in this worldwide fight against SARS-CoV-2. As we’ve all been affected in various ways by the...details. Our set of constructs comprises all SARS-CoV-2 viral proteins except for Nsp3 and Nsp16, and includes...is going on there!).  Find the Krogan Lab SARS-CoV-2 plasmids (Note: The Nsp6 construct is undergoing QC...

...interest in the cells you are studying. Read our SPARC blog post for more details. Figure 2: Dense (c... rising intracellular calcium levels within the cell. When levels of Ca2+ are high in the cell, calcineurin...infection (MOI) during cell library construction to ensure that the majority of cells receive one gRNA. The... of a genetically-defined subset of cells. By labeling fewer cells in a population, it’s easier to visualize...lengths, the density of cells that are labeled can be altered to dense (45% of cells labeled), intermediate...or aging cell populations. Figure 3: Micrographs of wild type (top panels) and cells with high...barcodes (iBARs) in their loop region. After infecting cells with sgRNAiBAR library containing lentiviral vectors...

...state-of-the-art methods of sex and gender analysis and 2) provides case studies with concrete examples of how...never considered the sex of their cells. For one thing, cells and cell lines degenerate over time—and sex... effects of using all female cells and tissues, specifically in stem cell therapies. Let’s go back to ...therapeutic capacity of stem cells. Yet, very few researchers consider the sex of their cells when doing experiments—which...sexes in basic research is excellent design), but they used all female stem cells—this was an unconscious... stem cells. Nor did they detect important differences in function between male and female cells. The ...experiment. In the case of stem cells, these factors may include cell type, the disease being treated,...

...change the media (add 2-3 mL of fresh media once a week), and do NOT harvest the cells before at least 10...titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more HEK293 cells and produce more rAdV ...infected cells show this “cytopathic effect.” Amplification can be repeated multiple times (2-4 rounds...creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the transgene...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...sequenced to confirm its integrity. Initial production (2-3 weeks) - Here you’ll produce the primary recombinant...rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification should result in a 10...

...safely. Recently, many SARS-CoV-2 researchers began pseudotyping the SARS-CoV-2 spike protein into a lower ... et a., 2018).   Figure 2: To pseudotype with the SARS-CoV-2 spike protein, scientists encode...of vesicular stomatitis virus, HIV-2, and human foamy virus in cell lines expressing galactosyl(alpha1...spike protein’s role in infecting cell lines without using intact SARS-CoV-2 virus. Scientists have been using...Particles Produced in Human Cells Are Inactivated by Human Serum. Molecular Therapy 2:218–222 . https://doi....production uses three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the packaging plasmid... pseudotyping methods with other viruses in a BSL-2 lab instead of a BSL-3 or BSL-4 lab. For more details...

... State University. Since its appearance, SARS-CoV-2 has spread to almost every part of the world manifesting...Rapid detection of 2019 novel coronavirus SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-...primers that target the E and the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences... method has been optimized to detect the SARS-CoV-2 virus directly using saliva without having to perform...and visual detection of novel coronavirus SARS-CoV-2 Sensitivity: 4.6-11 copies/microliter of RNA targets...single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification was...easy-to-deploy, protocol for Cas13-based detection of SARS-CoV-2 genetic material (CREST) Sensitivity: 10 copies of...

...cancer. These viruses need to be handled in a BSL-2 (or BSL-2+) lab and require additional safety measures.... BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL-2 materials, such...respiratory diseases in humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) ...recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus, many organizations...sprayed into the air. For lentivirus and other BSL-2 materials, you’ll need to use aerosol-tight caps, ...percentage of the decontaminate to be used for BSL-2 waste, and of course, if you are working with a substance...producer cell line, such as HEK-293T, production of viral particles, and transduction of a target cell line...

...the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed to...construct. For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required, but...Deliver your construct to your target cells. After recombination, exon 2 of the targeted gene will be removed... through an experiment designed to knock out exon 2 of a given gene.   Figure 1: A knockout targeting...the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted gene is disrupted (knocked out...recombination event will remove NeoR only and leave exon 2 floxed, as seen in line 4 of Figure 4. Since the loxP...recombined cells. Recombination is a rare event, so you must select as opposed to screen for cells where recombination...