...specificity in retinal cells. To do this, the group developed a new strategy for finding retinal cell-type ...monkey, and human retinas Next, the 113 AAV promoters that were active in mouse retinas were injected into...human retinas but not in mice was low (R=0.12), suggesting that mice could be used to eliminate promoters...gene expression in retinal cell types and classes in mice, primate, and human retinas. These synthetic ...types, such as inhibitory neurons, rods, cones, or retinal ganglion. Rather than using naturally occurring...they drove cell type specific expression in the retina.  Four approaches for designing synthetic promoters... with sequences upstream of the start codon of retinal cell-type specific genes from mice. Strategy #2...

...expression of other recombinases. Multiple orthogonal attB-attP pairs per recombinase may be arranged on...different recombinases are overlapping or nested on a register, then the operation of one recombinase on the... when treated with different combinations and orders of two recombinases. How might one read the state...they can control recombinase activity. For example, post-transcriptional recombinase activation via RNA...reduces the content of our paper, “Synthetic recombinase-based state machines in living cells” (1), into...explanation of how to adapt our engineered devices, recombinase-based state machines for your own experimental... about the order or timing of these factors. Recombinase-based state machines (RSMs), which take on a ...

...Having never been to China myself, I was pleased to be able to participate when the organizers of the...Dmitry Samarsky, and a very welcoming team from China’s Ribobio, the illustrious speaker list attracted...winners, one NIH Director and a co-founder of BGI-China presenting, it’s no wonder it was attended by over...were potential Addgene requestors and depositors. China is in one of the top 10 countries in number of plasmid...of Guangzhou and see some of the sites. Most fascinating to me was the outdoor market where one can shop...chemistry is complex and this work requires an interdisciplinary team.  Day two of the conference was kicked...from different species for genome engineering. The final keynote presentation was by Nobel Laureate Thomas...

...iGEM team originally used a guaB knockout E. coli strain to assay for functional decaffeination in cells...supplemented with various caffeinated beverages, but not with caffeine-free drinks. Finally, they demonstrated... pulse of blue light, and is reversible upon termination of light stimulation. CRY2clust enables faster... changes in intracellular Ca2+ levels than the original version containing CRY2 without the extra peptide..., termied soCoChR, by fusing a fragment of the kainate receptor subunit to the C-terminus of the protein...the group was able to demonstrate that while illumination of neighboring cells evokes action potentials...these APs are absent in soCoChR-expressing cells. Finally, using synaptic receptor blockers to distinguish...

...plasmid-based recombinant monoclonal antibodies Resources on Addgene.org Find recombinant antibody plasmids...leading to problems with reproducibility and an inability to improve upon these critical research tools.... Addgene to create a much needed open-access recombinant antibody/affinity reagent resource for the neuroscience...and data, the platform can be expanded for any recombinant antibody tool and therefore will grow to serve... the entire scientific community.     Why recombinant antibodies? Animal-based methods for antibody...cryopreservation (Bradbury & Plückthun, 2015a, b). Recombinant antibodies are plasmid-based and offer numerous...monoclonal antibodies. You can learn more about recombinant antibodies in this blog post. Hello, NABOR! ...

...Fvasconcellos.     How are Recombinant Antibodies Made? To make recombinant antibodies, you first need..., 2015).    The Many Benefits of Recombinant Antibodies Recombinant antibodies offer numerous advantages...definition of recombinant antibodies are essential for good reproducibility. Non-recombinant antibodies ...predict a similar revolution for antibodies. Recombinant antibody technology makes it easy for scientists...components of the scientific toolbox. What are Recombinant Antibodies? Antibodies are used by scientists...scientific institutions have proposed a shift to recombinant DNA-based antibody technologies (Bradbury & Plückthun...single antibody recognizing one epitope. (Bottom) Recombinant monoclonal antibodies are encoded in plasmids...

... multiple Addgenies and most recently updated by Ina Ersing in August 2023. Resources and references ...  Ina Ersing...absence of unexpected DNA contaminants. The data is also used to determine recombination rates in FLEX/DIO/fDIO...Endotoxin contamination in vector preparations can alter the immunogenic properties of the final product...mycoplasma contamination. To date, Addgene has never had a case of mycoplasma contamination. In the event...is performed on viral genomes isolated from the final AAV preparation. Sequencing results are analyzed...particularly in large animal studies. Endotoxin contamination is minimized by using an endotoxin-free plasmid...

..., Diana, Dolly, Eddy, Emma, Eric, Gizela, Henry, Ina, Inna, Ishrat, Jane, Jason, Jessica, Joel, Jonathan... revenue to build reserves so the company is sustainable. The difference is what nonprofits do with their..., Lianna, Luke, Marcy, Mary, Max, Maya, Meghan, Melina, Meron, Michael Lo., Michael Le., Michel, Michelle...

...By Susanna Bachle and Ina Ersing Seeding Labs and Addgene collaborate to provide scientific resources...behind Mycobacterium tuberculosis resistance to pyrazinamide (PZA) (a drug commonly used to treat tuberculosis... different approaches, including CRISPR and recombination-mediated genetic engineering (find more information...pathogenic Klebsiella and Pseudomonas isolates causing urinary tract infections. This project aims to examine ...Adeyi at the University of Ibadan. She works with medicinal plants such as Launaea taraxacifolia (Willd), ...pathophysiological complications of diabetes mellitus. Recombinant human proteins that play a role in insulin regulation...Ernesto Valdivia Silva is a master of interesting combinations: biomedicine and astrobiology. During the last...

...plasmids at Addgene! New from the viral service By Ina Ersing We regularly add new viral aliquots from our...protease used to cleave affinity tags from purified recombinant proteins at small and large scale.  This new .... Find the plasmids at Addgene! A new dual-deaminase base editor enables concurrent adenine and cytosine...Addgene New serotypes for the Cre-dependent Flpo recombinase pEF1a-DIO-FLPo-WPRE-hGHpA, which is now also ...

...series!  This post was originally written by Didem Goz Ayturk and updated by Ina Ersing in August 2023 ... your final stocks only contain rAAV. To minimize contamination, use appropriate decontamination procedures...40% PEG solution to each 100 mL of supernatant (final concentration of 8%). If, however, you’ve collected...digital PCR (ddPCR) (Lock et al., 2014), or determination of the median tissue culture infective dose ...primers targeting the viral genome or inverted terminal repeats (ITR). You should always have a high-quality...adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human Gene Therapy Methods, ...Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors [Preprint]. Molecular...

...experiment! Thanks to Addgenies Brian O’Neill and Ina Ersing for contributing their expertise! References...administered systemically, without causing disease? Recombinant adeno-associated viral vectors, or rAAVs, can...Laminin receptor (LR)3 AAV receptor2   AAV9 Terminal N-linked galactose3 AAV receptor2 Laminin ...found to transduce skeletal muscle, cardiac, and retinal cells (Issa et al., 2023). AAV1 capsids are available...including liver, muscle, lung, CNS, kidney, and retinal cells (Issa et al., 2023; Wu, Asokan, et al., 2006...cardiac, pancreas, smooth muscle, CNS, kidney, and retinal cells (Issa et al., 2023; Wu, Asokan, et al., 2006...transduce liver, skeletal muscle, pancreatic, and retinal cells (Issa et al., 2023). AAV9 packaging plasmids...

...) control of DNA recombination and gene expression can be advanced by the coordinated use of fellow SSR...system. The FLP recombinase recognizes FRT sites and initiates site directed gene recombination between those...channel under the control of Cre recombinase are used in combination with mouse lines that express Cre...regulated recombinase - keeping Cre out until needed But what can be done when the recombination should ...concentrations in combination with low concentrations of vectors expressing the required recombinase. The high...under the control of Flp recombinase together with inducible Cre recombinase (CreERT2), visualizes individual...Tamoxifen injection induces Cre-ERT2 recombinase, leading to recombination of the loxP sites and thereby to...

...components into a destination vector in a predefined order. Finally, the destination vectors are used ...positional groups. Eight final destination vectors, consisting of all four final RVD modules in each of ...promoter, N-terminal tag, coding sequence of the gene of interest, C-terminal tag, plant terminator, and plant...Gateway compatible destination vector for a maximum of 24 elements assembled into a final transgenesis construct...light-activated receptor tyrosine kinases Receptor tyrosine kinases (RTKs) are a large class of cell-surface...determined by competing recombination events at the genetic level. Upon Cre/loxP recombination, each transgene...:1852-61. A quicker method for protein inactivation Your protein of interest does x, but it also...

...models that express recombinases like Cre or Flp in their cells of interest and recombinase-dependent AAV vectors...Causes of recombination and leaky expression While leaky expression and spontaneous recombination is a known...sets of homologous recombination sites, they hypothesized that spontaneous recombination between these sites...even begins. This spontaneous recombination persists even in recombinase deficient E. coli and under conditions...vectors are much more prone to recombination and can exhibit recombination levels as high as 1-3%. In light...the recombination rate The first step in optimizing DIO/FLEx vectors is to know the recombination rate...system is off-target or spurious expression of recombinase-dependent AAV vectors. In this article, we will...

...against random recombinants.   Design your targeting construct. For recombination to occur in a ... for recombination, the negative selection marker selects against improper, random recombination into ...random recombination may incorporate the negative selection marker into the genome. This final product...After recombination has occurred, you'll first remove the resistance marker using Cre recombinase. Since...are 3 loxP sites, recombination can occur in multiple ways. The desired recombination event will remove...strategies to investigate a gene's function is to inactivate, or "knockout", the gene by replacing it or disrupting...yeast, mice, or Drosophila through homologous recombination. The concept is simple: deliver a template with...

...44632, the contaminant we mixed into the original AAV sample at 5%. Detecting 5% contamination is a good... Our recombination software looks for and quantifies the presence of recombinants in recombinase-dependent...sequences. Recombinase-dependent sequences may be subject to recombinase-independent recombination during ...containing the suspected contaminating sequence. In most cases, the source of the contamination can be identified...prep we suspect is contaminated. For AAV-68544, the best match for the contaminant is Addgene plasmid ...This recombination produces a minor pre-recombined population that does not require the recombinase for...viral vectors and estimate that recombinase-independent recombination occurs in 0.1-0.6% of AAV particles...

...human kinases remains challenging. Small molecule kinase inhibitors usually target soluble kinase domains...thereby rapidly create desired allele combinations and combinatorial genomic libraries. MAGE has allowed...pORTMAGE) expresses the λ Red recombinase enzymes, as well as a dominant-negative mutator allele of the... HA tagged BioID2 for N-terminal fusions and Myc tagged BioID2 for C-terminal fusions. Kim, et al. Mol... pCAG-T7-TALEN(Sangamo)-Destination series, pCAG-Golden-Gate-Esp3I-Destination Takashi Yamamoto pcDNA-TAL-NC2...Human kinase domain constructs for automated bacterial expression The misregulation of human kinases has...recently compiled the Human Kinase Domain Constructs Kit, a library of human kinase domain constructs developed...

... manipulation. Cre recombinase, originally named because it “causes recombination” (although later referred...cyclization recombinase”), is a 38 kDa protein responsible for intra- and inter-molecular recombination at the...alluded to above, the Cre recombinase catalyzes the site specific recombination event between two loxP sites...separate recombination events? One potential limitation of using loxP sites is the inability to tightly...specific recombination event in a single system. The Saccharomyces cerevisiae FLP-FRT recombination system... 1. Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. Sternberg, N. and.... 2. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Sauer...

...deposited by the Gradinaru lab. For simplicity, in each section we’ll list the Gradinaru capsids first, ...be used to target endothelial cells in the retina and spinal cord (Krolak et al., 2022). Systemic capsids... X., Luo, Y., Einarsson, P. H., Greenbaum, A., Jang, M. J., Deverman, B. E., & Gradinaru, V. (2020). Multiplexed...promoters and enhancers, miRNA target sites, and recombination target sequences, denoted by triangles] facilitate.... In cases where it’s critical to minimize or eliminate off-target expression, it may be better to choose...capsids in our repository are deposited by the Gradinaru lab. You can visit our Caltech Systemic Capsids...