...find both single-allelic and bi-allelic cells. Single-allelic cells usually make up the majority in culture...allows you to sort for GFP-positive cells and to enrich for those cells that were positively transfected....specificity. A good reference is this Cell paper: http://www.cell.com/abstract/S0092-8674(13)01015-5 As for.... A good example/reference is this Cell paper: http://www.cell.com/abstract/S0092-8674(13)01016-7 In the...CRISPR is used to engineer a locus, isolate single cell colonies, then genotype them, it is possible to ...or delivery the two plasmids together into your cells. If you want to use double nickase, you need to ...each well on 24-well plate, co-deliver into the cells with the Cas9/sgRNAs vector (like PX330). For large...

...organs, cells comprise compartments and structures, called organelles. Take a sneak peak inside a cell with...proteins, however, in this case the cell needs to be fixed and the cell membrane permeabilized in order for...Cooper GM. 2000. The Cell: A Molecular Approach. 2nd Edition. 3.  CureFFI.org. Cell Biology 04: The Secretory...involved in intracellular transport pathways, it is possible to map the internal organization of a cell. This...for labeling your subcellular structure of interest in the Allen Institute for Cell Science Plasmid Collection...with the images from the Allen Cell Explorer (1). When studying the function of a protein or its role in...biochemical methods thus removing the context of the cell. However, much knowledge about functionality can...

...can often be used in cells that are difficult to transfect, such as primary cells. Using RNPs can also...interest. This is commonly performed by transfecting cells with a plasmid, such as PX459, which encodes Cas9...must be considered when using this method:     Cells must be amenable to transfection or viral transduction...protein in complex with a targeting gRNA, to your cells of interest. Cas9 RNPs are capable of cleaving genomic...single or multi-gene knockouts in a wide variety of cell types, gene editing using homology directed repair...difficulties with protein expression that occur in cells where common eukaryotic promoters (such as CMV or...after transfection, and are quickly cleared from the cell via protein degradation pathways. This makes Cas9...

...endocrine cells. It follows that the specific targeting of these voltage indicators needs to be cell type ...Indicators As previously mentioned, cellular voltage is measured at the cell membrane, and this voltage is ...electrical events! Voltage indicators are voltmeters for cells! In this blog we will review how these indicators...related to the electrical impulse of the cardiac cells. To study this phenomenon you will likely want to...the voltage change across the membrane of cardiac cells. Similarly, neuronal signaling is carried out by... electrical charges propagated through multiple cells to do things like translate perceived signals to...Understanding neuronal pathways and the function of certain cells depends on the ability to monitor their signals ...

...incorporated into the edited genome. In mammalian cells such as stem cells, HDR is relatively rare and DSBs are predominantly...them into somatic cells affected by the disease and study what’s wrong with the cells. However, our excitement...sequence change the cell must undergo homology-directed repair (HDR), a distinct cellular DNA repair pathway...sequence-specific changes in human induced pluripotent stem cells (iPSCs). The CRISPR/Cas9 com­plex is very efficient... stranded breaks (DSBs) into genomic DNA in many cell types and often results in biallelic modifications... After genome editing with CRISPR/Cas9 in human cells was described in 2013 (Cong et al, Mai et al) we...excitement turned into frustration when we sequenced our cells and found how inefficiently our edits were being...

...K562 leukemia cell line and ~8 and 9% efficiencies in T-cells and hematopoietic stem cells respectively...transient transfection of 293T cells with helpers, and used to infect recipient cells. After G418 selection, ...using AAV vectors for gene editing in mammalian cells since 1998. In that year, Russell and Hirata from...used to routinely deliver sequences of interest to cells with efficiencies of 1-40% after G418 selection ...the p53 and PTEN tumor suppressor genes in human cells (Kim et al 2008). They have also used AAV gene editing...endogenous allele of the STAG2 tumor suppressor in human cells (Kim et al, 2016). The Waldman lab has designed ...modifying multiple genes simultaneously in the same cell, are more appropriate for a CRISPR-based approach...

...Time to expression AAVs have to infect cells, hijack host cell machinery, and synthesize a second strand...that can be used to express genes of interest in cell and animal models. AAVs are non-pathogenic, do not...be specifically targeted to a number of different cell types even when systemically administered in an ...ranging from transient gene expression in a particular cell type to CRISPR genome editing, optogenetics, and...application of AAV is delivery of genetic cargo to a host cell. In order to make room for a gene of interest, AAVs...allow the virus to infect (or not infect) different cell types with some specificity. There are a lot of ...ability of AAV to drive gene expression in specific cells and/or tissues is one of its biggest advantages....

...can actually infect cells. These assays typically involve infecting a target cell line with your virus...distinguish between a cell that has had one viral particle integrated into its genome and a cell that has had ...arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected and harvested...move ahead with your infection and make your stable cell line. While we’ve all experienced the pressure to...viral copies that have integrated into the target cell’s genome. Physical titer The two most common assays...functional titer uses FACS to count the number of target cells that are positive for vector encoded transgene expression...resistance genes carried on transfer plasmids. Target cells are transduced with serial dilutions of a viral ...

...proteins in ten ddifferent cell types, including difficult-to-manipulate primary cells, achieving knockdown ...stable cell lines overexpressing TRIM21, finding that TRIM21 overexpression did not affect cell proliferation...electroporated cells re-adhered to the culture plate within 4 hr of treatment. In some primary cell lines, endogenous...proliferation. These cells also did not contain changes in expression of TRIM21-associated genes, indicating...multiple methods for TRIM-Away delivery. For small cell populations, co-microinjection of TRIM21 protein...protein and the target antibody works well. In larger cell populations, you can deliver these components together...together using electroporation. In these bulk cell experiments, Clift et al. observed no stress or damage...

...modified zygotes or embryonic stem cells. Modified embryonic stem cells are injected into a blastocyst which...engineered cells. Similar to transgenic mice, knockout mice are engineered during the embryonic stem cell stage...not all cells in the blastocyst were successfully edited by the injection of embryonic stem cells. Also,...controls the cell types in which the conditional recombination can occur based on the cells in which Cre... interest into a zygote, or into embryonic stem cells that are then injected into a blastocyst. There ...of interest into a zygote or into embryonic stem cells. Non-homologous recombination incorporates this ...targeting vector is electroporated into embryonic stem cells and this will insert the gene at a specific site...