...Love your cells and they'll love you back A successful lentiviral infection begins with your cells, both ...both your packaging cells and your target cells. Poor tissue culture practices can lead to low titers and... promote cell growth. Overgrown cultures stimulate quiescence, the resting phase of the cell cycle; quiescent...quiescent cells do not take up nucleic acid or express transgenes as well as actively dividing cells resulting...reaching 108 cells per ml before the media becomes cloudy. Mycoplasma compete with host cells for nutrients...consumption due to the high cell density of the culture. In addition to benefitting your cells, medium exchanges...therefore titers obtained in one cell type may not translate well to other cell types. To overcome this issue...

...-dividing cells. The ability to infect non-dividing cells is not restricted to in vitro cell culture as...target cells. If you are transducing non-adherent cells you might need to centrifuge your cells with the...favourite gene into your favourite cell line or into primary cells, this blog article will give you some...infect both dividing and non-dividing cells, including stem cells. These properties make lentiviral vectors...immortalized cells lines but reach their limits when it comes to transferring DNA into primary cells or non-...non-dividing cells. To target these un-transfectable cells, labs use viral vectors and particularly retroviral...will first need to transfect producing cells, usually 293T cells, with your transfer plasmid and your packaging...

...With regard to target cells or organism, some primary cells and differentiated cell types are picky when...packaging plasmids into a viral production cell line, usually HEK-293 cells. When all of these viral plasmids ... rogue HEK-293 cell). This virus-containing media can then be placed on whatever cells you would like ...just zapping your cells? That really depends on two things. First, what type of cell or organism context...secreted into the cellular media, making viral collection fairly easy. Producer cell media can be collected...process of transferring foreign nucleic acid into a cell by a viral agent. Transductions are similar to transfections...processes which introduce foreign nucleic acid into cells by any means, but are unique in that they require...

...producing cells. After FACS, return the cells to doxycycline-free media and expand the cells. Use a subset...white inclusions in cells with GVs via phase contrast microscopy, GVs visible in cells via TEM, and purified... heterogeneity required single cell screening to isolate a clonal cell line capable of producing ultrasound...expression homogeneity, as the cells could be batch-sorted to isolate a polyclonal cell line with strong gas vesicle...transient transfection in rtTA+ cell lines (such as HEK293-TETOn cells), or they can also be integrated...PiggyBac transposase. In rtTA-cell lines, it is better to first engineer the cell line with rtTA, then integrate...own mARGAna cell line. (c) Z-projected phase contrast images of GV expressing MDA-MB-231 cells (top, scalebar...

...HEK293 cell culture and in live zebrafish. For cell culture experiments, only dL5**-expressing cells treated...inactivate target proteins or cells while having little effect on neighboring cells. The photosensitizer used...Here Light-induced cell ablation with MG-2I-dL5** He et al tested out light-induced cell ablation with MG...effective killing of dL5** cells without death of neighboring bystander wild-type cells.   Zebrafish experiments...fewer mCer3-labeled cells in their hearts 24 hours post treatment and no mCer3 cells present 96 hours post...increase cell ablation efficiency, but dL5** phototoxocity is induced when expressed on the cell surface...protein inactivation/cell ablation while having limited collateral damage to bystander cells. Are you ready ...

...gain access to some cells and tissues, such as human induced pluripotent stem cells (hiPSCs). Nanoblades... into particles at the cell membrane. Mature Nanoblades then bud into the cell culture supernatant and... Addgene. Genome editing primary cells with Nanoblades Primary cells are often refractory to gene delivery...mouse bone marrow (BM) cells, primary human hepatocytes, and hematopoietic stem cells (HSCs) using Nanoblades...-70% for these four cell types (Table 1). Additionally, hiPSCs and mouse BM cells still behaved like iPSCs...Primary cell type Gene targeted by guide Editing efficiency hiPSCs  EMX1  67% Mouse BM cells from GFP...mouse BM cells when using a gRNA targeting the Fto gene, while editing was not detected in cells electroporated...

...determine cell cycle length by examining the ratio of blue to red cells. Cells undergoing rapid cell cycles...’s difficult to observe the cell cycle in vivo, particularly if the cells are migratory or in deep tissues... BaF3 cells were transduced with H2B-FT virus and grown in varying concentrations of IL-3. Cells grown...proportion of blue fluorescence, indicating cell proliferation. BaF3 cells maintained in media without IL-3 show...H2B-FT consistently predicted the cell cycle speed across these different cell types. In one example, H2B-FT...expressed in murine B cells (BaF3), which depend on IL-3 to proliferate. The cells cultured with a low ...and had a greater number of red cells as measured by flow cytometry. Cells grown in high IL-3 showed mostly...

...Making contact with a cell Although an individual AAV particle’s ability to infect a cell is dependent on its...construction and the cell it’s infecting, getting these particles to infect cells is also very titer-dependent... particle to infect a cell, it has to come into physical contact with that cell. Since there is a limited...of surface area on each cell, the way to get more AAV in contact with the cell is to increase the AAV ...between the cell and any individual AAV particle is rare. But simply getting into the cell may not always...particles that have entered the cell now have to travel up the axon, through the cell, and potentially spread ...particles per cell, you may not be able to detect AAV-mediated gene expression in a target cell. Practical...

...levels in your cell lines, you can consider a cell treatment approach. If there is a cell treatment (e....select a candidate cell line (e.g. DepMap) – you’ll need to ensure that the parental cell lines of your chosen...the parental cell line, but not in the iso-genic knockout control. This can involve cell surface labeling...) compared to the WT cells (green histogram). The unfilled histograms show cells that have only been stained...the antibody’s labeling of the cell lines to the -omics data. The cell lines with higher expression of...the cell lines with lower expression should have less antibodies labeling them. We find that cell tracker... expression levels in the same cell lines/background. However, the cell treatment will frequently induce...

...infecting most cells, but not all. For example, LDLR expression in hematopoietic stem cells, T cells, and B cells... host cell. Some of these envelope-receptor pairings are broad, allowing delivery into many cell types...delivery to only a few cell types.  If you’re looking for more control in the cell types your lentiviral...protein causes cells that it infects to fuse together and form syncytias that cause the cells to die. Cytotoxicity...Cytotoxicity can also be cell type dependent: if a cell has few receptors for a specific envelope protein...lipoprotein receptors (LDLRs) on a cell surface to endocytose into the cell. The reason VSV-G is so commonly... cells is low. Infection of these cells with VSV-G pseudotyped virus requires the upregulation of LDLR...

...foreign antigens to adaptive immune cells, such as T cells and B cells. About a week following the initial...presenting cells. B cells in secondary lymphoid organs recognize specific antigens through the B cell receptor...have been described. NK Cell Signaling Natural killer (NK) cells can kill cells that have been infected... within an infected cell membrane, triggering cell death. TCR Signaling The T cell receptor (TCR) engages...through effects on numerous immune cell types including T cells and B cells. It also impacts the development...function of innate immune cells including NK cells, macrophages, dendritic cells, and granulocytes. TLR ...

...Non-Dividing Cells Transient Gene Expression Low Tropism for Mouse Cells and Hematopoietic Cells Low Packaging...fuses with the target cell’s membrane, allowing the viral particle access into the cell. The viral envelope... of cell the virus will target and fuse with. In nature, this determines which organisms and cells they...viruses can be engineered to be cell type specific. Once inside the host cell, the virus unleashes its cargo...wide range of cell types that are typically ‘difficult to transfect’, such as primary cells. They can also... them to easily deliver their “cargo” to target cells and organisms, and when that cargo is designed to...Viruses as biological tools In nature, viruses infect cells and organisms (which we refer to as hosts) via viral...

...infected cells, the other using wild-type Cas9 to remove the HIV-1 genome from infected cells. Read on...1 reservoirs HIV-1 infects cells in the immune system, notably CD4+ T-cells, and eventually leads to acquired...percentage of cells with activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that... genome from every infected cell and 2. prevent reinfection of those cells. Kaminski et al. designed gRNAs...Kaminski et al. observed no negative effects on cell viability, cell cycle progression, or apoptosis with Cas9...in HIV-1 infected T-cells to see if they could be rescued by CRISPR. CD4+ T-cells were isolated from healthy...1 is limited to a subset of T-cells, but it is unclear if enough T-cells could be reached to ablate a ...

...Winslow’s lab to label cells after cell-cell interactions. Using G-baToN, “sender” cells express GFP on the... and “receiver” cells express an anti-GFP nanobody on the cell surface. When the cells interact, GFP is...the receiver cell, and the receiver cell turns green. This system can be used in many cell types including...engineering with single-pot assembly Tracking cell-cell interactions New auxin inducible degron system....brgcm3sw     New system developed to track cell-cell interactions By Jennifer Tsang GFP-based Touching...including endothelial cell-pericyte, neuron-astrocyte, and cancer-stromal cell pairs.   Figure 2: Schematic...of the interaction between the sender cell and the receiver cell. Image from Tang et al., 2020.   ...

...Calcium phosphate Cells Transformed cell lines Transformed cell lines Transformed cell lines Cargo Nucleic...identify and enrich positive cells or a selection marker to generate stable cell lines. Transient or stable...and enrich positive cells. Likely contains a selection marker to generate stable cell lines. Packaging and...wide variety of mammalian cell lines. Useful for difficult to transfect cell types and can be used in ...protein are degraded within the cell.   Delivery methods for mammalian cell lines As mentioned above, the...components into your target cells. Delivery of these components into mammalian cell lines is quite broad and...Electroporation / Nucleofection Microinjection Cells Many types Single cells Cargo Nucleic acids or protein Nucleic...

...contaminating your transduction with the packaging cells. Packaging cell debris can be removed either by filtration... you can reduce the volume of cell culture medium on the packaging cells immediately following transfection...efficiency in cells that are sensitive to the toxicity of polybrene (e.g., hematopoietic or primary cells), and... the packaging cells after 1–3 days of selection. A lower percentage of surviving cells may be the result...adeno-associated virus transduction of cells." American Journal of Physiology-Cell Physiology 291.2 (2006): C386...vector, collecting virus, and transducing target cells, one may have the unfortunate experience of witnessing...witnessing…nothing. For some unclear reason, cells are not getting transduced. Here, I will share five hail...

... 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...high MOI, which is beneficial for a low number of cells and in vivo settings. Find the sgRNAiBAR library...

...expressed in your target cells. For easy-to-transfect cell types (e.g. HEK293 cells), transfection with standard... CRISPR machinery. For more difficult cells (e.g. primary cells), viral delivery of CRISPR reagents may...identify and enrich positive cells, or selection marker to generate stable cell lines (for details, see Plasmids...wide variety of mammalian cell lines Useful for difficult to transfect cell types, can also be used in...possible genotypes within the resulting cell population. Some cells may be wild type due to either (1) a ...efficient target cleavage in cells expressing both Cas9 and gRNA. Edited cells may be homozygous or heterozygous...will use the example of CRISPR/Cas9 in mammalian cells, many of these principles apply to using CRISPR ...

...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... 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,...the most commonly used cell lines appearing in American Journal of Physiology-Cell Physiology over the past...aware of in our checklist for tissue and cell research. Using cells from both sexes ensures that the diagnostics...

...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...tool that allows for the labeling of a subset of cells with the same genetic makeup, overcomes all of these...