...Darrell Kotton is the director of the Center for Regenerative Medicine at Boston University Medical School... Darrell recently joined the Addgene Board of Directors where he hopes to help keep us focused on our ...cell-based therapies underway. Chapter 7: Generating entire lungs using a decellurized scaffold as a base. ...Darrell's goals as a member of the Addgene board of directors - increasing open source sharing and helping us...Investigator in the Kotton Laboratory and is the founding Director of the Center for Regenerative Medicine of Boston...

...for cells that had successfully incorporated the desired mutation, and then derive mice from these engineered...performance. In its natural setting, CRISPR-Cas9 is an acquired immune system in bacteria and archaea. As you ...changes can be made in the genome via the homology directed repair pathway (HDR), be it a single nucleotide... so efficient that you can inject the reagents directly into fertilized mouse eggs, circumventing the ...CRISPR is much more efficient at generating the desired mutant offspring, with 85% success compared to ...CRISPR/Cas9 Genome Editing Method that Does Not Require Ex Vivo Handling of Embryos. Current Protocols ...

...homology directed repair template to facilitate the selection of cells containing your desired edit. The...Addgene is proud to announce that we recently acquired the ability to distribute plasmids with the piggyBac...using the piggyBac™ transposase, which facilitates direct integration of the donor-transposon (carrying your...23723351. PubMed Central PMCID: PMC3690869. 5. D. Grégoire and M. Kmita, “Recombination between inverted ...Engineering with FLEx Vectors  Learn how Homology Directed Repair Is Used for Genome Editing Resources on...

...the Cole Lab selected promoters for cell types directly linked to disease, including motor neurons (-3mnx1...PhoCl LSSmCherry1 & RDSmCherry1: Engineering and directed evolution of new RFP variants The toolbox of fluorescent...compatible with two-photon microscopy using Ti-Sapphire lasers. pBAD-RDSmCherry1 is a red-shifted variant...Read the group’s PLOS ONE paper for more on the directed evolution of mCherry and the spectral properties...identified as non-essential. The knockouts replace entire protein-coding regions (excluding start and stop...individual strain includes a xylose-inducible dCas9 paired with a single, unique, and constitutively expressed...localization at telomeres and found that TZAP binds directly to TTAGGG repeats via its zinc finger domains....

...it may not be productive to apply for jobs that require a bachelor’s degree if you have a PhD. 4) Customize...even excitement, in a position. Everyone prefers to hire someone that will be excited to be there. If you...resume pdf document. 6) Use your network! During the entire process, use whatever means you can to get your...the hiring manager on paper or as an email send directly from a mutual connection. If the organization ...organization is large, you will need to be more directed and target someone in a specific department and...

...relatives suffering from similar diseases. Others were inspired by the imminent threat of climate change to study...interesting questions and your excitement will in turn inspire the people around you. Be proactive Rather than...assistants), about their experiences. Research requires dedication and effort, so building a strong understanding... interests and that you want to travel in new directions. During my grad school interviews, one of the...advice Give yourself permission to pursue your own desired path. Careers in science are also not limited to...industry, so feel free to explore! Addgene has an entire page dedicated to different career options from...

...a need for the exclusive ligation of fully base-paired nicks with no gaps. For example, DNA assembly methods...Gibson Assembly® and NEBuilder® HiFi DNA Assembly, require nick-selective ligases, such as Taq DNA Ligase,...ligases generally prefer fully Watson-Crick base-paired dsDNA substrates to those containing one or more...Tm around the mismatch) over annealed fully base-paired substrates. Thus, high fidelity ligation can be...Walter G. Verly. "Nicks 3′ or 5′ to AP sites or to mispaired bases, and one-nucleotide gaps can be sealed by...method using a LIF dual‐channel detection system for direct identification of allelic composition of mutated...

...behavioral applications where drawn out effects are required. Targeted manipulation Both optogenetic and chemogenetic...chemogenetics on the Addgene website. Optogenetics also requires that light of the correct wavelength reach receptors...such as zebrafish larvae. Mouse studies, however, require a permanent intracranial implant to be surgically...deschloroclozapine (DCZ) has been developed to meet these requirements. TL;DR If precise spatiotemporal control of...cells expressing receptors Invasiveness Mice require permanent intracranial implant Noninvasive Controlling...

...recognize using an RNA guide. Non-target strand: The paired strand of foreign DNA cut by Cas9, whose sequence...labs have created Cas9 enzymes with looser PAM requirements, which are available from Addgene!   RNA crRNA... repeat section of the crRNA, forming the repeat:antirepeat duplex. tracrRNAs also contain multiple stem...Cas proteins, like LbCas12a or AsCas12a, do not require a tracrRNA. sgRNA: “Single-guide RNAs” were invented... of an RNA guide (either crRNA or sgRNA). Cas9 requires perfect complementarity in this region between...

...certain threshold, it becomes depolarized and will fire a signal. The non-specific cation channel ChR2 and... becomes more negative it becomes less likely to fire and is said to be hyperpolarized. The chloride pump...peak absorption wavelengths and light intensity requirements of different opsins, studies have shown that...only Kolmer-Agduhr cells on one side of the body required photostimulation in order to drive the full symmetrical... (2013): 119-126. PubMed PMID: 23246238. Wyart, Claire, et al. "Optogenetic dissection of a behavioural...

...have the required expertise to help move your project forward? Most projects typically require a diverse...brainstorming sessions to maximize productivity and direction of the project? What is your timeline? A typical...design, execute, analyze, repeat pathway that is required of scientists.” Professor George Church encourages...approach. At Addgene, we've heard of labs where new hires spend the first 3 months reading and writing a proposal...

...antibody that is directly conjugated to your reporter circumvents the issue. However, directly conjugated primary...Strategies for avoiding background secondary binding Direct immunohistochemistry (Figure 2A)  How do you avoid...species on species IHC. (A) One solution is to use direct IHC, which uses just a primary antibody that has...has your reporter (in this case a fluorophore) directly attached to it. (B) Another solution is to combine...

...can change entire culture) Specific requirements for hiring more common Likelier to hire on general...implicit aspects of today’s postdoctoral training can directly interfere with a seamless jump into industry.”... opportunities to do science related jobs that require no bench work Better resources available ($)—can...

...showing that this protein could cleave DNA in vitro, directed by phosphorylated DNA guides. Unfortunately, Swarts...mammalian cells, likely due to high temperature requirements. To solve this problem, Gao et al. searched ...Like Cas9, NgAgo is also able to mediate homology-directed repair when a template is supplied. Find Plasmids...Cas9 and the other CRISPR enzymes, NgAgo does not require a PAM sequence, so users should have much more ...single mismatch at any position of the DNA guide impaired NgAgo activity, with three mismatches abolishing...

... that we are doing just that. Do you like the direction the blog has taken over the past year? Do you ... 10 blog posts of 2015 1. CRISPR 101: Homology Directed Repair Addgenie Chari Cortez explains some of ...of the basic science behind homology directed repair (HDR) and how it’s used in CRISPR genome editing. ...vector contaminants in your cloning experiments and tired of having to PCR many colonies to find a successful...making a transition into a new job or maybe to an entirely new career path, a career coach might be able ...

...multiple genes. Modifying the CRISPRi system for multidirectional control The CRISPR interference (CRISPRi) ...Modifying the CRISPRi system for more flexible multidirectional control, a new study repurposes the sgRNA ...regulators allows a greater degree of control of desired expression levels dependent upon the type of transcriptional... E. coli. The utility of simultaneous and multidirectional multiplex control via scRNA design is readily...RNAs can be designed to allow simultaneous, multidirectional and tunable control of gene expression. ...

...species. Administration of these dyes results in direct binding between the dye and the endogenous target...target, and thus no genetically encoded tag is required. These dyes can also be attached to an antibody ...substrate ligand motif attached to a fluorophore. When paired with a protein of interest, these tools provide...biotechnology (Grimm & Lavis, 2022). These variations directly impact the way you can utilize these dyes. The...another factor to consider. If your application requires longer periods of exposure, you’ll want a more...intermolecular FRET for ligand-receptor interactions, require specific considerations regarding the choice of...

...Drobizhev et al. 2011). But this requires other lasers than the standard Ti:Sapphire laser. It is also important...processes during embryonic development. These are required for a growing organism to make the complicated...for many years, researchers lacked good tools to directly follow immune cells in lymphoid organs. The development...Good two-photon probes when using a standard Ti:Sapphire Laser include: Blue-green fluorescent proteins...PMCID: PMC2700268. 8. Hickman, Heather D., et al. "Direct priming of antiviral CD8+ T cells in the peripheral...

...of the fittest. Cancer cells find a way to grow haywire, access and create more vasculature to feed themselves... tumor cells that escape the elimination stage acquire genetic alterations and alter cell-surface antigen...cells would normally kill cancer cells but are directed not to through these various signals. Over-production...the microenvironment help create tolerance beyond direct proximity to the tumor; chemokine-aided activation...a variety of up and coming therapies. Future directions and open questions in cancer treatment We are... is to be successful, cancer immunotherapy must aspire to be a field of healthcare as dynamic as cancer...

... event. What are exosome AAVs? In 2012, Casey Maguire and colleagues from Massachusetts General Hospital...that are released from cells during production (Maguire et al., 2012). They saw that these AAV particles...development of the exo-AAV isolation protocol by Maguire and colleagues, other researchers have tried to...delivery of therapeutic proteins to the brain which requires crossing the blood-brain-barrier. In summary, ...: 28082074. PubMed Central PMCID: PMC5368844.  Maguire, Casey A., et al. "Microvesicle-associated AAV ...