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...to cross mice to create double knockout lines and Cre-lox lines, and how to properly control for genetically...goal is to generate mice that have two modified genes in their genome, and assuming Mendalian patterns...your parental mice were all homozygous for your genes of interest, your F1 mice will be carriers at those...mice that are homozygous for all of your desired genes. Remember Punnett squares from high school biology... that harbor homozygous alleles at both of your genes of interest. About 1 in 4 mice will be homozygous...the promoter being used should be tested in cell lines to determine how it impacts the expression level...

...regulate the transcription of specific genes. What if one of those genes coded for a protein that regulated...combinations of different candidate genes at once to identify the best cocktail of genes when the relationship between...Level 0: Cloning of basic parts (promoters, UTRs, genes, tags, etc.) into MoClo-compatible vectors. Level...the DNA that encodes the protein itself or other genes within the circuit. These interactions yield a cellular...stacking is the use of a combination of multiple genes to achieve a desired effect. This is commonly used...

...right CRISPR vectors on Addgene and found instead ones that were… pretty close, or at least close enough...Fragmid depositor John Doench reports even the greenest members of his lab, high school interns, were ...created over five months. When they tested those clones (in duplicate), 93% had the correct banding pattern...enzyme digest. They sent 82 of the correctly banded clones for plasmid sequencing and found 98% of the plasmids...Drosophilia Plasmid pRDB_051 (for stable fly cell lines; attL and attR sequences for genome integration ...Drosophilia Plasmid pRDB_052 (for stable fly cell lines; Hygromycin resistance gene; for genome integration...own Fragmid components! Ensure your backbone and genes contain only the Bbsl cut sites needed for the Fragmid...

...ex: rods or cones) or cell classes (ex: photoreceptors which encompassed both rods and cones).  Targeted...of cell types, such as inhibitory neurons, rods, cones, or retinal ganglion. Rather than using naturally...of the start codon of retinal cell-type specific genes from mice. Strategy #2 (ProB): Create promoters ... transcription initiation sites of at least two genes with high expression and high cell-type specificity...to loosely match the specificity of their source genes, i.e. if a synthetic promoter expressed in cells... Transgenic animals allow for the expression of genes in particular tissues, but they are costly and time-consuming...

...sequencing of puromycin resistant clones, they found that 80% and 50% of clones displayed proper insertion at... Addgene depositor Takashi Yamamoto’s lab has harnessed MMEJ to create a new method for CRISPR gene knock-in.... Using their PITCh plasmids, GFP knock-in cell lines can be created in about a month and a half, without...promoter, and the technique can be adapted to other transgenes. It’s important to note that the fluorescence... would be advantageous to adapt PITCh to insert genes into AAVS1, the “safe harbor locus” of the human...and Ling Lu. (2016). “Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus...

...organisms that can't reproduce with wild ones. For example, Drosophila lines with compound autosomes are completely...populations. Whereas most genomic changes impose a fitness cost and are eliminated by natural selection, gene...didn't work quite as well as anticipated4, greater awareness will certainly improve safety. What safeguards...? There are ongoing efforts to develop formal guidelines to answer this question. Until then, using multiple...guest blogger! An inventor of technologies that harness evolution, Kevin Esvelt studies ways of using molecular...www.sculptingevolution.org. References: Burt, A. Site-specific selfish genes as tools for the control and genetic engineering...

...has always been focused on gene transfer, from transgenes to plants, chloroplast to the nucleus, or pathogens...The MoClo Plant Kit makes it really easy to clone genes for expression in plant cells. Sylvester and I chose...includes everyone’s favorite promoters, plus a few new ones that are not used so much but have comparable expression...and give similar stability to transcripts as the ones that people use all the time. Now there are many...to a lot of pathogens. It’s also easy to express genes in, which makes it a good laboratory workhorse. ...Tim-Martin Ehnert, Stefan Werner, Jonathan D. G. Jones, Nicola J. Patron, and Sylvestre Marillonnet. ACS...Plant SynBio Reading: Quick, Versatile Plant Transgenesis with GreenGate Plasmids  ...

...insertions, deletions, and point mutagenesis to complex assembly of multiple genes. Primer design for each type... to the 5’ end of the reverse primer. Mutagenesis - Mutagenesis is performed by designing primers astride...included in a single PCR, for example for subcloning genes from different templates. All PCRs are run as an...@jakefwatson and @JGarciaNafria.  References 1. Jones, Douglas H., and Bruce H. Howard. "A rapid method...method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase...

...bacteria to multiply, spreading their resistance genes throughout the bacterial population. But what if...them to multiply and propagate their resistance genes. Our society is hoping to combat antibiotic resistance.... Above all, we need new antimicrobials, ideally ones for which the development of resistance will be .... targeted beta-lactam and quinolone resistance genes in E. coli using plasmid and phagemid delivery systems...therapy. Another strategy for CRISPR antimicrobials combines them with both replication-competent phages and...work targeted two distinct beta-lactam resistance genes, again with very high specificity. This double phage...prevents sensitive bacteria from acquiring resistance genes. Yosef et al. suggest that it could be valuable ...

...sequence of interest can lead to poor expression of genes within that sequence and should therefore be prevented... recombinase are used in combination with mouse lines that express Cre only in the cell type of interest...and expression of a separate gene. In these mouse lines, gene knockout can be induced by Cre recombinase...researcher exquisite control over expression of multiple genes in specific cells at specific times. Many thanks...at MIT and is fascinated by the question of how genes and proteins coordinate billions of neurons to form...Properties of FLP Recombinase Evolved by Cycling Mutagenesis.” Nature biotechnology 16(7): 657–62. PubMed ...Deleter Mouse in C57Bl/6 Based on FLPo Recombinase.” Genesis 48(8): 512–20. PubMed PMID: 20506501. 6. Logie,...

...Yang, a professor in synthetic biology from the Chinese Academy of Sciences. In the Yang lab, we’re continually... consists of one plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing...Fortunately, we were able to successfully delete genes of interest in BL21(DE3) using the newly generated...with multiple terminators can serve to interrupt genes (or gene clusters) in metabolic engineering. So ...a strain library with 1-11 copies of integrated genes. We obtained a stable and high-yielding strain which...guest bloggers! Qi Li obtained her Ph.D. from the Chinese Academy of Sciences (CAS) Center for Excellence...Ph.D. from Shanghai Institute of Biochemistry, Chinese Academy of Sciences. He joined Shanghai Institutes...

...their test genes, the researchers at the BIOFAB showed that expression of many different genes was predictable...coli. 800 and 1900 Series If you’re expressing two genes from a single transcript, or if you want to prevent...create the pCell-Free vectors, Gateway-compatible backbones that enable cell-free expression of proteins in... constructed a library of eGFP-tagged human ORF clones to allow testing and comparison of different cell-free...Cell-free expression test kit represents a set of 88 clones in a pCellFree vector that enables protein expression...enzyme function can lead to surprisingly large fitness effects during adaptive evolution of antibiotic...

... enhanced GFP (EGFP), a variant with improved brightness (Tsien, 1998). Another mutation (A206K) is necessary...solution to avoid autofluorescence is offered by bioluminescent proteins. These probes generate light by a ...there is no need to excite the sample. Although bioluminescent probes are usually dim relative to fluorescent...recent engineering efforts have increased the brightness of these genetically encoded light emitters (...palette of Nano-lanterns for real-time multicolor luminescence imaging." Proceedings of the National Academy...PMC4394297. 16. Iwano, Satoshi, et al. "Single-cell bioluminescence imaging of deep tissue in freely moving animals...the Fluorescent Protein Collection Find empty backbones for fluorescent protein fusions Browse the Fluorescent...

...were able to introduce the desired mutations in genes associated with dementia, such as APP and PSEN1,...when screening reasonable amounts of single cell clones. Furthermore, CRISPR/Cas9 editing is often bi-allelic...2016). For those of you involved in the tedious business of iPSC clone picking, a 100-fold decrease is ...the difference between picking 100 versus 10,000 clones! We would therefore strongly encourage any researcher...intended sequence change and screen a few hundred clones to find the right one Step 2 – Edit the cells a...steps, you’ll be screening a total of a few hundred clones - Still better than 10.000, right? (Kwart, Paquet...incorporate mutations without screening thousands of clones to find a positive one. So if you have better things...

... discriminate between positive and negative clones. Clones with insertion will show only linearized plasmid...: Can you summarize HR template vector design guidelines? A7: A few notes below are considerations for...plasmid of ~8.5 kb (only AgeI will be able to cut). Clones without insertion will show a ~1 kb and ~7.5 kb...) on each colony in order to screen for positive ones.  -- OR -- If your HR template has a selection... primers that may work better than the published ones, so if/when you use HercII or Kapa Hifi the reaction...During Gene Targeting with Zinc-Finger Nucleases. G3 Genes Genomes Genetics, 3(4), 657–664. https://doi.org...generation of mice carrying mutations in multiple genes by CRISPR/CAS-Mediated Genome Engineering. Cell,...

...lifecycle. Halloween genes The Halloween genes are a set of cytochrome P450 genes discovered in Drosophila...Mutations in these genes are lethal, associated with defective exoskeleton formations. These genes were appropriately...1983): 813-816. Gilbert, Lawrence I. "Halloween genes encode P450 enzymes that mediate steroid hormone...

...takes several weeks to delete or deplete individual genes. Michael Laub’s lab developed a CRISPRi system to...downregulate expression of specific Caulobacter genes using a catalytically dead Cas9 (dCas9) derived ...Streptococcus thermophilus (the popular Streptococcus pyogenes system does not work well in Caulobacter). To ...system can also be used to downregulate multiple genes at once. Read the paper in mBio Find the CRISPRi...transgene design and delivery. They generated hPSC lines with AAVS1-integrated, inducible, and fluorescent...which increases activation of methylation-silenced genes. Read the paper in Nature Communications Find Casilio-ME...

...lentiviral backbones designed right here at Addgene. Here's what makes these new backbones special: Strong...deactivation rates Addgene's own empty lentiviral backbones New recombinant antibodies for Glypicans DNA editing...deactivation). Both have significantly improved brightness, kinetics, and sensitivity. iGluSnFR4f is well-suited... Build your way: Explore Addgene's new empty backbones By Meghan Rego You asked, we listened! One of ...favorite features and limitations of existing backbones, we're excited to share our very own set of empty...labeling By Alyssa Shepard To fully understand how genes are regulated, it's important to know what proteins...

... so genes. It would be great if we could just go in and quickly manipulate individual mouse genes and ...genome editing system derived from Streptococcus pyogenes. An alternative Cas9, from Streptococcus aureus...effective at editing mouse embryos as Cas9 from S. pyogenes and has the advantage of being smaller (Zhang ...C57BL/6, for which we have germline competent ESC lines. Second, the process is much quicker. It takes 3...Wang is a professor at the Institute of Zoology, Chinese Academy of Sciences, and he is interested in using...Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell ...

...challenge when researchers are communicating across disciplines or doing different tasks. But these challenges...can be exaggerated when communicating across time zones, digitally, and asynchronously. The best way to ...collaborating with remotely. Depending on the willingness of the team to adopt various tools, this may ...collaboration includes scientists working in different time zones, it’s important that everyone can access data and... collaboration. Some things to figure out are timelines for experiments and manuscripts, who is responsible...your recurring meetings and reaching out if any milestones are missed. Spend time in person Presenting...

...thanks to our guest writer, Pamela J. Hines, PhD! Pamela J. Hines is a Senior Editor at Science magazine... post was contributed by guest writer Pamela J. Hines PhD, Senior Editor at Science Magazine. Although...replication and gene transcription during vertebrate embryogenesis. My interests, though, have proven unruly. Instead...journalism? Take a look at our news editors. A fondness for visualization of science? Look at our graphics...French horn, and rhubarb. Follow her on Twitter @Pam_Hines and LinkedIn. Additional Resources on the Addgene...

...graduate student, you are in a system that, quite honestly, does not value your potential contribution to... science, you are likely lacking the necessary business background and/or connections to even begin this...pitch what you hope to build.  Then ask for their honest opinions -- this part might hurt, but if you pitch...made, we considered the feedback and adjusted our business model accordingly until we had a model that was...This will allow you to discuss each project and business decision before making it, to have an internal...faculty, venture capital and angel investors, and business development heads at major companies.  The majority...

...signaling is likely to have). Anterograde tracing outlines neurons from their cell bodies to the terminals...terminals of their axons; while retrograde tracing outlines neurons in the opposite direction, from the terminals... dynein, while anterograde transport relies on kinesin. Different rates of speed for various forms of ...predominately anterograde tracing, biotinylated dextran amines (Veenman et al., 1992) or fluorescent molecules... achieved by using optogenetically controlled transgenes, in combination with conditional transgenic mice... M., and Schnell, M. (2008). Concepts in the pathogenesis of rabies. Future Virol 3(5), 481–490. PubMed...Ho, A., Nicholas, E., Hirokawa, K.E., Bohn, P., Joines, K.M., Peng, H., Hawrylycz, M.J., Phillips, J.W...

...MSCV (Murine Stem Cell Virus) sequences. Packaging genes (viral Gag-Pol): Gag is a structural precursor protein...tropisms.  Packaging using helper-free packaging cell lines This method utilizes a packaging cell line that ...and/or Env, eliminating the need to deliver these genes in trans by specific plasmids. This method, therefore...standard γ-retroviruses use the Gag, Pol, and Env genes for packaging; however, they are different viruses...replication incompetent? Yes, as with lentiviruses, the genes required for γ-retroviral packaging and transduction...element; sequence that stimulates the expression of transgenes via increased nuclear export. LTR Subcomponents...

...nucleic acid-based vaccines. These vectors provide several advantages over traditional vaccines, including 1... knock it out, etc. It’s how we understand what genes do! Viruses have several options to allow you to...engineered to introduce frameshift mutations in genes of interest, turning expression off permanently....2007.   Clinical applications Viruses in vaccines helped pave the way for their use in other clinical...The use of viral vectors in vaccine development. Vaccines. 7, 75 (2022). DOI: https://doi.org/10.1038/s41541...

...virus will target and fuse with. In nature, this determines which organisms and cells they infect. In the...the fear of random genomic integration causing mutagenesis in the host. Adenoviruses can deliver relatively...Dividing Cells Moderate Immunogenicity Insertional Mutagenesis Possible Lentivirus Infects non-dividing...non-dividing cells Insertional Mutagenesis Possible Adenovirus Remains Episomal (Little Risk of...of Insertional Mutagenesis) High Gene Expression Infects Non-Dividing Cells Large Packaging Capacity (... AAV Remains Episomal (Little Risk of Mutagenesis) Very Low Immunogenicity Infects Non-Dividing...

...all known genes. This is a perfect example of an unbiased, reverse screen – all known genes are assayed... a biological process. Players can be proteins, genes, enzymes, drugs, or anything else that plays a role...where there is removal of known factors (such as a genes) and the loss of those factors is assessed. The ...similar to CRISPR, but instead of knocking out the genes of interest (or the whole genome), they knock down...especially useful if you are interested in studying genes whose knockout would be lethal to cells and thus...

...more commonly used backbones and specific primers you can use to confirm your clones. You can also find...screen will help you more easily identify successful clones so you have to weed through fewer colonies after...antibiotic resistance. But what about identifying clones that contain your desired insert? This is where...how you can use restriction digest to screen your clones, but there are many other ways to choose restriction...PCR.   Sanger sequencing Sanger sequencing determines the precise order of nucleotides within a DNA...

...on cell lines susceptible to or dependent on certain media components, and are rescued by genes supplied...colonies (used to complement auxotrophic cell lines), or genes which rescue a lethal phenotype. Resources... transformed plasmid. Such plasmids may contain genes that allow for the use of a particular substrate...list of Bacterial Expression Systems Find Empty Backbones for Your Experiments    ...

...number of genes involved in cardiovascular and metabolic diseases. We are knocking out genes that have...mutant versions of 15 different disease-associated genes. Addgene spoke to Musunuru about how the new kit...kit is quite useful in generating the mutant cell lines that one needs to do disease modeling in stem cells...several mutations in the same gene or in multiple genes, all of which seem to contribute to a particular...iPS) cells from patients and try to compare those lines, they may all have the mutations but they will also...

...producing landslide after landslide, only adding more stones to the field. Fortunately, our bodies have been... (Th) cells. These cells produce inflammatory cytokines - interleukin (IL)-12 and interferon gamma (IFNγ... promotes the production of VEGF to increase angiogenesis around the tumor. This in turn regulates the...cancer. Surgical removal causes increases in tumor cytokines like TGFβ and IL-6. However, when strengthened...response. Antibodies targeting the tumor-augmenting cytokines produced in the tumor microenvironment TGFβ, IL...necroptotic tumor cells have also been tested as vaccines and therapeutics. Their uptake by DCs is increased...marker blockade and suppression of tumor-aiding cytokines, for example, will more broadly activate the immune...

...are tumor microenvironments made of?  A tumor’s genesis is divided into 3 phases: initiation, progression...active regions, microenvironments shape a tumor’s evasiveness, evolution, and response to treatment. Some microenvironment...transport miRNA from drug-resistant cells to sensitive ones. Though this process is not fully understood, a ...formation by inhibiting cell growth, invasion, and angiogenesis (Allinen et al., 2004). In fact, Sanford H. ...influence tumor microenvironments: immunology and angiogenesis (Witz et al., 2009). Then, scientists began ...Fernando Schmitt. "Microenvironment in breast tumorigenesis: Friend or foe?." Histology and histopathology...

...experiments. Test dTAG molecule toxicity in new cell lines Treat parental cells (or cells expressing a control...FKBP12F36V-fusion chimera of interest. In the cell lines we have evaluated to date, we observed little to...FKBP12F36V fusion chimera degradation. In the cell lines we have evaluated to date, we have not observed ...was employed to evaluate degradation of ENL. Cell lines were engineered to express ENL-FKBP12F36V and endogenous... employed to evaluate degradation of MELK. Cell lines were engineered to express FKBP12F36V-MELK and endogenous...was employed to evaluate degradation of YY1. Cell lines were engineered to express YY1-FKBP12F36V using ...

...either through a chromogenic or chemiluminescent reaction. Chemiluminescence, which is light produced by ...the primary amine groups from lysines, sulfhydryl groups from cysteines, or carbohydrates from glycosylation... found throughout the antibody. 2) Endogenous cysteines, such as those that make up the disulfide bonds...antibody (Mueller, et al., 1988). If some of those lysines are in the antigen binding region or even just ...

...expression yielded editing of 80–90% in human cell lines and primary cells. Although cell entry of a Cas9...minimal toxicity, including in clinically-relevant lines such as CAR-T cells. Plus, this method is fast! ...break up the dimerization without losing that brightness and stability. Here’s a quick overview of these...variants:   Figure 2: Inner mitochondrial membranes imaged with structured illumination microscopy.... This variant has similar photostability and brightness compared to the original, though its maturation...versions. Thanks to Christophe Leterrier, whose FocalPlanes piece on mStayGold inspired and informed this...

... to, ones targeting muscular dystrophy, cardiovascular disease, inflammatory diseases, blindness, and ... cancer treatment by traditional gene therapy pipelines (viral), and researchers are currently working...many more in the earlier stages of the pipeline, ones that have experienced hiccups (either clinically...challenges with therapies involve patient safety, effectiveness, and delivery. Patient safety is important for...concerning when using a systemic approach. Regarding effectiveness, if the editing frequency isn’t high enough ...

...stripping to remove, so start with the weaker binding ones first to preserve your sample. Pro tip! Because ...reprobing works best with western blots that use chemiluminescent or fluorescent detection, like horseradish...protein retention. Following this step, most PVDF membranes recommend re-wetting with methanol before rinsing...the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed with caution. Unlike PVDF...your own stripping solution using a recipe like the ones shared by Bio-Techne, ThermoFisher, or Millipore...remains, whether that involves adding fresh chemiluminescent substrate or imaging your fluorescent probe...

...into ‘split-vector systems’ by separating viral genes and transgene expression across several plasmids...that allow functional screening of thousands of genes in genome-wide experiments. Gamma-retroviral vs...direct the cellular RNAi machinery to silence target genes. This empty MuLV vector from the Weinberg lab enables...find more lentiviral expression vectors (empty backbones) here. Cloning and expressing new shRNA sequences...lentiviral backbone from the Zhang lab expressing S. pyogenes Cas9 and a gRNA cassette. It can be used to produce...studies. The ability to simultaneously alter multiple genes via infection with a single ecotropic or amphotropic...

...So how can you use CRISPR to introduce large FP genes into C. elegans? By using a split FP approach. Split-wrmScarlet... fluoresce. The team engineered C. elegans cell lines to express wrmScarlet1-10 unattached to any other... validated this tool by targeting six different genes whose protein products have distinct localizations... library that targets human glycosyltransferase genes and other proteins related to cellular glycosylation...deletes human alternative cassette exons and disrupts genes with alternative exons. Get the library from Addgene... format! The library inhibits over 18,000 human genes. Find the lentiviral preps at Addgene hSyn1-SIO-stGtACR1...

... constructs for tagging the kinesins KIF5A and KIF5B as well as backbones for generating other SNAPf-,...the conformational dynamics of the motor protein kinesin-1, the Twelvetrees Lab took advantage of the self-labeling... will be useful not only for future studies of kinesins but for many other proteins of interest too! Find....A.S., Craggs, T.D., & Twelvetrees A.E. (2024) Kinesin-1 is highly flexible and adopts an open conformation...mouse monoclonal antibodies targeting the human genes and are recommended for use in immunohistochemistry...