...NGG-3′ (“N” can be any nucleotide base, followed by 2 guanines). This limits the use of ABEs as DNA sites... in the promoter region of gamma hemoglobin (HGB1/2) leads to continuous expression of fetal hemoglobin...primary human cells  Cell therapy approaches can use base editors to correct mutations in stem cells and return...primary human cell editing, the researchers tested ABE8 in CD34+ hematopoietic stem cells. ABE8 efficiently... In addition to stem cells the authors targeted 6 genes in primary human T cells using 8 selected ABE8s...to edit primary human T cells efficiently makes it a promising tool for T cell therapy development.  Reducing...be introduced into cells as a plasmid or as mRNA constructs. In primary human cells mRNA delivery of ABEs...

...gene modification. Maeder ML et al. Mol Cell. . 2008. Jul 25. 31(2):294-301. PubMed PMID 18657511 . Oligomerized...OPEN OPEN gGTCATCTGCcattcGAGGCGGACa REST corepressor 2 OZ553 and OZ554 OPEN OPEN gCTCACCATCtgttggaTGCGATGGAa...

...Regulation Ben Deverman 2:00 PM Cre-DOG and Combinatorial Strategies Connie Cepko 2:45 PM Break 3:00 PM (...GCaMP6f-P2A-nls-dTomato Detailed protocol: AAV Production in HEK293 Cells Please share materials by uploading to your Dropbox...

...PMC5118044. 2. Kim, Y., et al., The Generation of Human Induced Pluripotent Stem Cells from Blood Cells: An Efficient...blood are reprogrammed immediately after collection [2]. PBMCs display unique epigenetic signatures that ...human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells, 2013. 31(3): p. 458...possible to reprogram differentiated cells into induced pluripotent stem cells (iPSCs) by the addition of reprogramming... somatic cells that are otherwise difficult to obtain. These can then be used to perform cell therapy ...in vitro. Find stem cell plasmids at Addgene Many sources of adult somatic cells have been probed for...Using Serial Plating of Reprogrammed Cells by Centrifugation. Stem Cells Int, 2016. 2016: p. 1329459. PubMed...

...Haploid Embryonic Stem Cells Carrying a Guide RNA Library. Cell Stem Cell . 17 (2), 221–232. PMID: 26165924...used to transduce your experimental cell population (Figure 8E). In a 2-vector system, you’ll need to either...of CRISPR-Cas9 with Bacteriophage Proteins. Cell , 168 (1–2), 150-158.e10. PMID: 28041849 Sakuma, T., Nishikawa... regulation of transcription in eukaryotes. Cell . 154 (2), 442–451. PMID: 23849981 Gilbert, L. A., Horlbeck...Transcriptional Programs with CRISPR RNA Scaffolds. Cell . 160 (1–2), 339–350. PMID: 25533786 Cas9 alternatives...single RNA-Guided endonuclease of a Class 2 CRISPR-CAS system. Cell , 163 (3), 759–771. PMID: 26422227 Zetsche...left free to interact with the target DNA. Figure 2: Overview of the NHEJ repair mechanism Cas9 will only...

...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...

...referred to as intermolecular or bimolecular FRET) or (2) conformational changes within a protein where the...standard consisting of ECFP and EYFP connected by 2 GGSGGS repeats pET28CLY3 Peptide linker standard consisting...Protein Resources: Empty Backbones Biosensors Subcellular localization Optogenetics Background Förster ... Constructs to target mTurquoise2 to various subcellular compartments pCEP4CyPet-MAMM Cyan Mammalian Expresses...

...), 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...

...Constitutive 1, 2, 5, 9 Deisseroth 26973 pAAV-hSyn-hChR2(H134R)-EYFP Syn ChR2/H134R EYFP Constitutive 1, 2, 5, 9....WPRE.SV40 CAG ChR2/H134R mCherry Constitutive 1, 2, 5, 9 Deisseroth 127090 pAAV-CAG-DIO-ChR2(H134R)-eYFP...Optogenetics AAV Optogenetic tools can be used to control cell activity in living tissue with light. Optogenetics...

..., Nat Biotech 2007 ) in mammalian cells or zebrafish). pMG10 (a '“2-in-1” ZFN cassette for expression ...vectors designed to express nucleases in different cell types. Zinc finger arrays can be transferred in-...vectors designed to express nucleases in different cell types. Zinc finger arrays created using ZFC reagents...nuclease domains: pST1374 (for expression in mammalian cells or zebrafish) pDW1775 (for expression in plants)... Miller et al., Nat Biotech 2007 ) in mammalian cells or zebrafish (can be used as a substitute for pMLM335... Miller et al., Nat Biotech 2007 ) in mammalian cells or zebrafish (can be used as a substitute for pMLM336... Miller et al., Nat Biotech 2007 ) in mammalian cells or zebrafish). pMLM802 (for expression of a ZFN ...

...(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...

...the use of pAdEasy-2 can increase the capacity of the adenoviral vector. pAdEasy-2 does not contain the...Adenovirus packaging plasmid that lacks E1 and E3 pAdEasy-2 Adenovirus packaging plasmid that lacks E1, E3, and...compatible with the pAdEasy-2 packaging plasmid; Supplies viral protein E4 E1 Supplied in trans by 293...AdEasier-1 cells BJ5183 E. coli cells containing the pAdEasy-1 packaging plasmid 911E4 cells Cell line compatible...then transformed into AdEasier-1 cells , which are BJ5183 E. coli cells containing pAdEasy™. pAdEasy™ is... 293/911 cells throughout virus production. During experiments, GFP can be used to sort cells infected...must be transfected into 911E4 cells for virus production, as 293 cells do not contain E4. Adenovirus ...

...surface of most immature B cells; function is unknown Monomer with a valency of 2 IgE Protects against parasites...such as anaphylactic shock Monomer with a valency of 2 IgG The most abundant isotype in blood, also found...secondary immune response Monomer with a valency of 2 IgG1; IgG2; IgG3; IgG4 IgM Produced during the primary... ranging from antibody fragments (shown in Figure 2) to completely unrelated molecules. Examples include...used for assembling protein nanostructures. Figure 2: An antibody (IgG, far left) compared to alternative... produced. Cells - Antibodies can also be produced in cell cultures using hybridomas, cell cultures created...created by fusing B cells with myeloma cells (immortalized B cells). The genetic reorganization that creates...

...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme... time and are potentially heritable in dividing cells. CRISPR Activate and Interfere technologies also...

...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...

...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...

...gene-specific CRISPR and donor vectors as well as pX330S-2-PITCh, expressing a generic gRNA targeting the PITCh...Protocols, 2016 . Yamamoto Tagging Plasmids: pX330S-2-PITCh - expresses Cas9 & PITCh-gRNA pX330A-FBL/PITCh...Drosophila cells. N terminal tagging in Drosophila cells 3.2 MB C terminal tagging in Drosophila cells 3.3 MB...following article: Natsume, et al. Cell Reports 2016 Förstemann Drosophila Cell Tagging System The Förstemann... Allen Institute for Cell Science Plasmid Collection The Allen Institute for Cell Science has produced...tagged, human, induced pluripotent stem cell (hiPSC) lines. These cell lines were created using CRISPR and...tagging transcription factors with FLAG in human cell lines. To repeat the tagging in your system, use...

...AAV vectors for trial purposes in serotypes AAV1, 2, 5, 8, 9 and retrograde Retrograde AAV To target projection... tools that effectively transduce many mammalian cell types. AAV is used in a wide range of experiments...Tetracycline Transactivators, Affinity Purification, Cell Ablation Monosynaptic Neuronal Tracing Rabies Virus-Based...

...optogenetic tool is the widely used Channelrhodopsin-2 (ChR2) from Chlamydomonas reinhardtii . ChR2 is a ... optical switch tools to measure and manipulate cells and their biomolecular processes. Plasmid...to blue light and allows cations to flow into the cell. Variants of channelrhodopsin have been engineered...improve the expression and membrane targeting in host cells. Other microbial opsins, such as the Halorhodopsins...actively move cations or anions into or out of the cell. When using these opsins as a tool to control neuronal...plasmids that use light to control a protein or cellular activity. For example, search "Cdc42", "transcription...

...nonspecific cation influx into the cell when stimulated. Channelrhodopsin-2 (ChR2), the first widely adopted... (Phy) interaction 660 Cryptochromes Cryptochrome 2 and CIB1 Blue-light–mediated induction of protein ...interactions based on Arabidopsis thaliana cryptochrome 2 and CIB1 450 LARIAT Inhibits protein function by reversibly...customizable TALE DNA-binding domain with cryptochrome 2 protein and its interacting partner CIB1 to control...transcription effectors OPTOSTIM PHR domain2 of cryptochrome 2 (Cry2) from Arabidopsis thaliana is fused to truncated... with single-spike temporal resolution. PLoS One. 2(3):e299. PMID 17375185 Kim B, Lin MZ. 2013. Optobiology...comparative analysis of microbial opsins. Nat Methods. 9(2):159-72. PMID 22179551 Saunders A, Johnson CA, Sabatini...