...GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...resultant JEDI-2P sensors overcome many challenges in 2-photon in-vivo imaging of voltage fluctuations. Image...suitable for in vivo optogenetic use.   Fig. 2: A) HcKCR1 is highly selective for K+ over other Na...slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies...

...Part 2: Building Community  My editor’s choice pick for this month is “Grad School Advice Part 2: Building...Posts from October 2016: Sequencing Options for CRISPR Genotyping Grad School Advice Part 2: Building ...truly is dressed as Unikitty from The Lego Movie). October was no less busy for the Addgene blog which once.... Of course there were many fantastic posts in October. Click on the links below to catch up on ways to...

...products have distinct localizations. Figure 2: Split-wrmScarlet labeling proteins with different...

...ask new and interesting questions in your own lab. 2. Seek multiple mentors during your postdoc At the ... of your postdoctoral training experience. If you're going to commit to doing a postdoctoral fellowship...contributed by Erik Snapp, the Director of Student and Postdoctoral Programs at theJanelia Research Campus of the...the Howard Hughes Medical Institute. Today's postdoctoral fellows (PFs) face a number of challenges ranging...academia - the main reason most people enter postdoctoral training. Similarly, there are several factors...1. Reverse engineer success On Day 1 of your postdoctoral experience, define what will be needed to achieve...you searched for and eventually identified a postdoctoral mentor. A good mentor typically writes the grants...

...Vale RD. Cell. 2014 Oct 23;159(3):635-46. doi: 10.1016/j.cell.2014.09.039. Epub 2014 Oct 9. Pubmed. Find...resolved loci with physical map distances of 75 and 2 Mbp, with the calculated fluorescent distances correlating...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the...

... Biol. 2011 Jan 14;405(2):315-24. doi: 10.1016/j.jmb.2010.10.038. Epub 2010 Oct 28. PubMed. Find the ...;3(11):820-31. doi: 10.1021/sb500273n. Epub 2014 Oct 11. PubMed. Find the plasmids from this publication...Marcotte EM, Voigt CA. Nature. 2005 Nov 24;438(7067):441-2. PubMed. Find the plasmids from this publication ...

...Weissman JS. Cell. 2014 Oct 23;159(3):647-61. doi: 10.1016/j.cell.2014.09.029. Epub 2014 Oct 9.     ...VP64 fusion; 2. A sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop 2; 3. The MS2... the top 10 hits.” Two-way CRISPR screening In October, Jonathan Weissman’s lab introduced additional ...

...Retroviruses and cancer. Rev Infect Dis. 1982 Sep-Oct;4(5):1015-25. DOI: 10.1093/clinids/4.5.1015 Lee C.S...Application of Viruses to Biotechnology. Viruses. 2021 Oct 14;13(10):2073. DOI: 10.3390/v13102073 Resources ...Era of Personalized Medicine. Genes Dis. 2017 Jun;4(2):43-63. DOI: 10.1016/j.gendis.2017.04.001 Maetzig...

...multidrug resistance-1 gene (pfmdr1). J Parasitol. 2011 Oct;97(5):939-42. doi: 10.1645/GE-2792.1. Epub 2011 May...-87. doi: 10.1016/j.mimet.2005.09.007. Epub 2005 Oct 10. PMID: 16216354 ...mean you have one copy of the gene; an RQ value of 2 means you have two copies, and so on and so forth....quantitative PCR. Expert Rev Mol Diagn. 2005 Mar;5(2):209-19. doi: 10.1586/14737159.5.2.209. PMID: 15833050...

...Plasmids and Chromosomes. Microbiol Spectr. 2014 Oct;2(5). doi: 10.1128/microbiolspec.PLAS-0025-2014. PMID...identification.   Fig. 2: Gel image of Plasmid 61564: pBAMD1-2 (~4.7 kb) digested with SpeI and...factor E-1 from Proteus mirabilis. Biochemistry. 1968 Oct;7(10):3513-20. doi: 10.1021/bi00850a028. PMID: 4878700... at ~3.6 kb and ~1.1 kb (lane 3). Uncut DNA (lane 2) has a band at <3 kb, which corresponds to the supercoiled...formation of plasmid multimers. Cell. 1976 Dec;9(4 PT 2):707-16. doi: 10.1016/0092-8674(76)90134-3. PMID: ...dimers in Escherichia coli K-12. Plasmid. 1989 Mar;21(2):138-41. doi: 10.1016/0147-619x(89)90057-7. PMID: ...Behavior of Plasmid DNA. Microbiol Spectr. 2015 Apr;3(2):10.1128/microbiolspec.PLAS-0036-2014. doi: 10.1128...

... determines fetal hemoglobin level. Science 2013 Oct 11;342(6155):253-257. PubMed. 3. Canver MC, Bauer...with TALENs, in collaboration with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore...nucleases. Genome Res. 2010 Jan;20(1):81-89. PubMed. 2. Bauer DE, Kamran SC, Lessard S, Xu J, Fujiwara Y,...

...Ward JD, Reiner DJ, Goldstein B. Nat Methods. 2013 Oct;10(10):1028-34. doi: 10.1038/nmeth.2641. Epub 2013...one injection step! The entire protocol takes about 2-3 weeks but requires less than eight hours worth of...for C. elegans genome modification. Figure 2: Schematic from Dickinson et al. (2015) illustrating...

...CcdB.” Biotechniques. 1996 Aug;21(2):320-3. PubMed PMID: 8862819. 2. Bahassi, EM., et al. “F plasmid CcdB...using the F plasmid ccdB killer gene”. Gene. 1994 Oct 11;148(1):71-4. PubMed PMID: 7926841. 4. Bernard,... desired insert should not grow.   Figure 2: Image from http://teachline.ls.huji.ac.il/72682/tutorials...

...self-associating fluorescent domains. ACS Chem Biol. 2013 Oct 18;8(10):2133-9. doi: 10.1021/cb400427b. Epub 2013...MagFRET: The First Genetically Encoded Fluorescent Mg(2+) Sensor. PLoS One. 2013 Dec 2;8(12):e82009. doi: ...

...U, Takahashi M, Gage FH, Verma IM. J Virol. 1998 Oct;72(10):8150-7. PubMed PMID: 9733856. PubMed Central...create the 2nd-generation lentiviral system (Figure 2). The HIV genes that do remain are very important ...1996 Apr 12;272(5259):263-7. PubMed PMID: 8602510. 2. A third-generation lentivirus vector with a conditional...CH, Humeau LM, Rebello T. J Gene Med. 2013 Feb;15(2):78-82. PubMed PMID: 23322669. 6. Genetic treatment... Banasik MB, McCray PB Jr. Gene Ther. 2010 Feb;17(2):150-7. PubMed PMID: 19847206. Additional Resources...

...PubMed Central PMCID: PMC3640491. 2. Grimm, D., et al. "Titration of AAV-2 particles via a novel capsid ELISA...PCR versus analog real-time PCR. Nat Methods. 2013 Oct;10(10):1003-5. PubMed PMID: 22035192. PubMed Central...report the physical titer of a viral solution (Figure 2). Because physical titers are used for dosing purposes...mean and how they can be compared.   Figure 2: Label that comes on Addgene’s AAV aliquots. Our titer...of genomes can limit production of recombinant AAV-2." Gene therapy 6.7 (1999). PubMed PMID: 10455443. ...Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material." Human gene therapy 21.10...

...Last updated on Oct 14, 2020 by Seth Kasowitz. This post was contributed by the gene editing team at ...this year. Designing the donor plasmid Figure 2: Donor plasmid design strategy. A donor plasmid contains...

..., detection, and control. Int J Pharm. 2018 Oct 25;550(1-2):251-268. doi: 10.1016/j.ijpharm.2018.08.043...and particle formation. J Pharm Sci. 2015 Feb;104(2):602-11. doi: 10.1002/jps.24259. Epub 2014 Nov 21....

... van Rheenen J, Simons BD, Clevers H. Cell. 2010 Oct 1;143(1):134-44. PubMed. Drosophila Brainbow: a recombinase-based...Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow 3.2. ...M-CFP expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate... each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for example, Brainbow...processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...

...Currently available from the Kim lab are 2 presynaptic and 2 postsynaptic targeting mGRASP plasmids. Additionally...transgenesis constructs. Kirchmaier et al., PLOS One 2013 Oct 7;8(10):e76117. Irreversible peptide-peptide ligation...9(5):1104-12. Heinemann et al., FEBS Lett 2012 Oct 19;586(20):3716-22. Park et al., Science 2011 Aug... cells were pulsed with 2µg/ml of Doxycycline for 2 hours, and imaged on the Keyence BZ-9000 fluorescence...work! Robinson et al., Dev Cell 2010 Feb 16;18(2):324-31. Cheeseman et al., J Cell Sci 2013 May 1...promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated... of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly...

... Multiple Introns.” Plant Communications, vol. 2, no. 2, Elsevier BV, Mar. 2021, p. 100135. Crossref, ... Technologies. Cold Spring Harbor Laboratory, 27 Oct. 2023. Crossref, https://doi.org/10.1016/j.xgen.2024.100519...Constructs.” PLoS ONE, edited by Jean Peccoud, vol. 6, no. 2, Public Library of Science (PLoS), 18 Feb. 2011, p... vol. 5, no. 10, American Chemical Society (ACS), 2 May 2016, pp. 1059–69. Crossref, https://doi.org/10.1021...of Plant Genomes.” The Plant Genome, vol. 16, no. 2, Wiley, 9 Mar. 2023. Crossref, https://doi.org/10.1002...Crossref, https://doi.org/10.1186/s12870-020-02388-2. Stuttmann, Johannes, et al. “Highly Efficient Multiplex...Crossref, https://doi.org/10.1038/s41598-019-46171-2. Resources on Addgene.org Modular Cloning (MoClo...

...Last updated on Oct 1, 2020 by Aliyah Weinstein. This post was contributed by guest bloggers, Wenning...posts: Part 1: Genetically Engineered Mice and Part 2: Breeding and Crossing Mice. My first CRISPR mouse...is less predictable. In our best case, we saw that 2 out of 3 mice carried a 5 kb insertion in the ROSA...

...them in Regenerative Medicine. Acta Naturae, 2010. 2(2): p. 18-28. PubMed PMID: 22649638. PubMed Central...capability to differentiate into specialized cell types [2]. We general speak of 4 levels of stem cell potency...zygote through to the morula stage are totipotent [2-4]. Pluripotency: When a cell has the ability to differentiate...stem cells (ESCs), which I discuss in detail below [2]. Multipotency: When a cell can differentiate into...PMID: 26726264. PubMed Central PMCID: PMC4686367. 2. Jaenisch, R. and R. Young, Stem cells, the molecular... the ectopic expression of four embryonic genes- OCT4, SOX2, KLF4, and C-MYC. iPSCs were first generated...communication and publishing after completing her postdoctoral training from Massachusetts General Hospital...

...resulting from non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation...each sample can be quickly calculated (details in [2]) and can be used to quantify rates of mutation in... have been reported for mismatch nuclease assays (2, 6, 7). Ability to distinguish between mono-allelic...assays, in addition to testing their performance (2). Remember that although digital PCR is a great screening...PMID: 24509632. PubMed Central PMCID: PMC4063274. 2. Findlay, S. D., Vincent, K. M., Berman, J. R. & Postovit...This post was contributed by Scott Findlay, a Postdoctoral Fellow at the University of Alberta. If you’... out of frame! Scott Findlay is currently a Postdoctoral Fellow at the University of Alberta. He is interested...

... this problem? (2-3 sentences) What will be the focus of your first grant? (1-2 sentences) ...of scientific success is publications and grants. 2. Write succinct and easy to understand documents A...consideration. 3. Present an easy to follow seminar See item 2. Attendees will include grad students (that are solicited...people would agree is important to understand. (1-2 sentences). For example: Distinguishing correctly ... potential collaborations, critiques of your postdoctoral research, etc. The second question is whether...from Oregon Health Sciences University, did his postdoctoral fellowship with Jennifer Lippincott-Schwartz...and now serves as the Director of Student and Postdoctoral Programs at the Janelia Research Campus of the...

...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of...for generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...understanding the mechanisms of reprogramming, and 2) studies with clinical endpoints. In the first scenario...PubMed PMID: 27764162. PubMed Central PMCID: 5072705. 2. Sommer, Cesar A., and Gustavo Mostoslavsky. "The ...challenges." Journal of Cellular Physiology 228, no. 2 (2012): 267-75. PubMed PMID: 22767332.  3. Kim, Dohoon...PubMed PMID: 23546745. PubMed Central PMCID: 4176696. 2. Yusa, Kosuke, Roland Rad, Junji Takeda, and Allan...generation. Expert opinion on biological therapy, 10 2, 231-42. PubMed PMID: 20088717. PubMed Central PMCID...

... non-physiological, and difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several...Biol, 2003. 163(2): p. 257-69. PubMed PMID: 14581454. PubMed Central PMCID: PMC2173526. 2. Zacharias, D.... nature are prone to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [3]....form dimers in a cell. The Kd of EGFP is 0.11 mM [2]. Following the simple logic outlined above, you might...organized smooth endoplasmic reticulum (OSER) whorls. 2)A dimerizing protein of interest fused to a dimerizing... designed to detect resulting in false positives [2, 4]. Solutions to the polymer problem There are some...Citrine) can be monomerized with an A206K mutation [2]. Addgene has many of these constructs and they are...

...your target gene! Tanenbaum et al., Cell. 2014 Oct 8. pii: S0092-8674(14)01227-6. Multi-bit genetic... Zetsche et al., Nat Biotechnol. 2015 Feb 2;33(2):139-42. doi: 10.1038/nbt.3149. Light-inducible ...and on your favourite signalling pathways (Figure 2). They are all available now at Addgene. 1 Lungu...20;19(4):507-17. 10.1016/j.chembiol.2012.02.006. 2 Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; ...Nihongaki et al., Chemistry and Biology. 2015 Feb 19; 22(2):169-74. High efficiency cloning for enzyme screening...inducible, gene knockdown with shRNA or shRNA-miR30, (2) tamoxifen-inducible CreERT2 gene deletion, (3) simultaneous...different fluorescent proteins, 8 constitutive promoters, 2 includible promoters, 3 polyA terminators and various...

...Quotes taken directly from participants (summarized in 2) touched upon many problems in this area: “ [The] ...PMID: 24733905. Pubmed Central PMCID: PMC4000813. 2. McDowell, Gary S., et al. "Shaping the Future of ...A fair deal for PhD students and postdocs." Elife 2 (2013): e01139. Pubmed PMID: 24137543. Pubmed Central...the Future of Research will be held from 22-24 October. This blog has been contributed by guest blogger...strong supporter of increasing the number of postdoctoral scientists at University of Maryland. Paula... can register here or live stream the event on October 22nd and 23rd here. Many thanks to our guest blogger...

...mechanism of action are presented in Figure 2. Figure 2: Single-stranded donor DNA interaction with...Nature biotechnology (2016). PubMed PMID: 26789497.  2. Lin, Steven, et al. "Enhanced homology-directed human...contributed by guest blogger Chris Richardson, a Postdoctoral Researcher in Jacob Corn’s lab. CRISPR-Cas9 ... optional donor DNA. Chris Richardson is a Postdoctoral Researcher in Jacob Corn’s lab investigating...

...the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed to... through an experiment designed to knock out exon 2 of a given gene.   Figure 1: A knockout targeting...For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required, but 6 to 14 kb...construct to your target cells. After recombination, exon 2 of the targeted gene will be removed from the chromosome...the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted gene is disrupted (knocked out...recombination event will remove NeoR only and leave exon 2 floxed, as seen in line 4 of Figure 4. Since the loxP... protein with GFP, as seen in blue flame plasmid OCT4-eGFP-PGK-Puro from the Jaenisch lab.   Figure...

...properly exiting the ER and fluorescing (see Figure 2) [5].  When using FPs containing two cysteine residues...compartments including the secretory pathway (Figure 2), the inner membrane space of mitochondria and chloroplasts...PMID: 18228466. Pubmed Central PMCID: PMC2875081. 2. Snapp, Erik Lee. "Fluorescent proteins: a cell biologist's...from Oregon Health Sciences University, did his postdoctoral fellowship with Jennifer Lippincott-Schwartz... from Albert Einstein College of Medicine. Her doctoral research was conducted in the laboratory of Erik...subcellular environment. Currently, she is a SPIRE postdoctoral fellow at the University of North Carolina at...

...NGG)  Editing window ~2 nt  85175  pBK-EE-BE3  SpCas9 (NGG)  Editing window ~2 nt  85176  pBK-YE2-...quantum yield of 0.54) and mScarlet-H with M164H (2-fold improvement in photostability compared to mScarlet...SpCas9 base editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated...pBK-YE2-BE3  SpCas9 (NGG)  Editing window ~2 nt  85177  pBK-YEE-BE3  SpCas9 (NGG)  Lower on-target activity...activity than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity...Ladders Are Awesome Tools: They are comprised of 2 plasmids, pPSU1 and pPSU2, that can be amplified affordably...libraries The Gross Lab at UCSF recently deposited 2 Bacillus subtilis libraries with Addgene – the B. ...

...Topic) 0:00 - 2:00 : Introduction to the podcast from producers Tyler Ford and Eddy Page. 2:00 - 3:55 : ...MD PhD process and explains why he loved the first 2 years of medical school. 7:05 - 10:55 : Alex's focus...Pathology at Masachusetts General Hospital and Postdoctoral Fellow in the Church and Collins labs at the...

...different steps and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail,...stimulation         In the video above, Channelrhodopsin 2 (Chr2) has been expressed in the motor cortex of a...lasers are relatively cheap and will run you about $2-3,000/laser. However, an important caveat is that ... the light-gated cation channel, Channelrhodopsin 2 (ChR2), exists in several states: closed/light receptive...PMID: 21628464; PubMed Central PMCID: PMC3138296. 2. Sparta DR, et al. Construction of implantable optical...board, Jacqueline N Crawley  [et al]. 2012;Chapter 2:Unit2 16. doi: 10.1002/0471142301.ns0216s58. PubMed...University of Michigan and he is currently a Postdoctoral Fellow in the lab of Mary Jeanne Kreek at the...

...revert back to a pluripotent stage (iPSC formation) [2, 3]. The iPSCs then proliferate and redifferentiate... more common delivery methods include retrovirus [2], lentivirus [4], adenovirus [5], Sendai virus [6]...2006. 441(7097): p. 1061-7. PubMed PMID: 16810240. 2. Takahashi, K., et al., Induction of pluripotent stem... Pluripotent Stem Cells. Cell Stem Cell, 2015. 17(2): p. 178-94. PubMed PMID: 26189426. 15. Si-Tayeb, ...induced pluripotent stem cells. Stem Cell Rev, 2015. 11(2): p. 242-53. PubMed PMID: 25578634. PubMed Central... to regeneration. Nat Rev Mol Cell Biol, 2011. 12(2): p. 79-89. PubMed PMID: 21252997. 37. Merrell, A....involves overexpression of four reprogramming factors- OCT4, SOX2, KLF4, and C-MYC - that induce a differentiated...

...phosphorylation events in the proteomes of higher mammals (2). How can we begin to understand the importance of... the open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...optimized Sep-OTSλ and can recommend its use (6).  2. C321, a specialized bacterial host strain for pSer-incorporation...biology4.5 (2002): E127-E130. PubMed PMID: 11988757. 2. Hornbeck, Peter V., et al. "PhosphoSitePlus, 2014...contributed by guest blogger Natalie Niemi, a postdoctoral fellow at the Morgridge Institute for Research...blogger Natalie Niemi! Natalie Niemi, Ph.D., is a postdoctoral fellow at the Morgridge Institute for Research...

...post-doctoral fellow at Vanderbilt University. She studies beta-cell function and failure in Type 2 Diabetes...scientists have 1) the courage to make mistakes and 2) the integrity to recognize these mistakes quickly...available to people who want to join the dialog and 2) remember that science should not be partisan: everyone...chair of the postdoctoral engagement committee and active member of the university's Postdoctoral Association...in the veracity of its work. Alina Chan is a postdoctoral researcher at Harvard Medical School working...runs hand in hand with policy. Alexandra is a postdoctoral researcher in the University of Michigan Department...continue the dialogue. Erin Golden is currently a postdoctoral fellow at the University of Colorado Anschutz...

... go back and finish the review.  Peer-review step 2: Detailed assessment After this initial assessment...salt-induced changes in root-to-shoot ratio in Arabidopsis, (2) expression patterns in plants with enhanced sodium...post was contributed by Magdalena Julkowska, a postdoctoral researcher at KAUST, Saudi Arabia. From the... submission to the subsequent journal.  As a postdoctoral researcher studying plant development and abiotic...

...that increase cancelled. Rounding out the remainder, 2% of postdocs who weren’t going to get a salary increase...with their plans, and 17 have cancelled their plans. 2 of those raising - the University of Illinois and ...of Information Act (FOIA) requests for their postdoctoral salary information as of December 1st. Salary...are an important step forward in helping the postdoctoral population continue to pursue academic work,...of the scientific enterprise by reducing the postdoctoral population which keeps growing despite a lack...