...enhances iPSC generation as does replacing C-MYC with L-MYC [10, 11]. While the mechanisms underlying ...addition of an shRNA for P53 and replacing C-MYC with L-MYC in the episomal reprogramming cocktail by ...Nakagawa, M., et al., Promotion of direct reprogramming by transformation-deficient Myc. Proc Natl Acad Sci... reprogramming factors- OCT3/4, SOX2, KLF4, and C-MYC [1]. For the derivation of clinical grade hiPSCs... that encoded six factors - OCT3/4, SOX2, KLF4, C-MYC, LIN28 and SV40 large T antigen (SV40LT). The method...the site of insertion and which can potentially promote tumorigenesis in vivo. The phenomenon has been ...pCXLE-hSK 27078 SOX2 and KLF4 pCXLE-hUL 27080 L-MYC and LIN28 pCXLE-hOCT3/4-shp53-F 27077 OCT3/4 and...

...genome-wide. It was designed based on MYC-ChIP-seq data from several MYC-dependent cancer cell lines. It can...selection marker, Thy1.1, in addition to puromycin resistance.  The MYC-CRISPR library targets E-boxes genome-wide...optimized their initial system by using a stronger promoter and different biochemical secondary energy sources... can be applied for identification of essential MYC binding sites in a wide range of human cells.  A new... system is capable of robust expression of high G+C content genes, as validated through monitoring the...’s lab previously developed such a system in Streptomyces, a prokaryotic genus capable of producing a ...

...Zhi Ying Chen, Robert C. Robbins, Mark A. Kay, Michael T. Longaker, and Joseph C. Wu. "A nonviral minicircle... viral particles that express oncogenes, such as Myc. Viral vectors also have the largest genomic footprint...transgenes is a concern as well, and reactivation of Myc or other oncogenes after differentiation has been...expresses multiple reprogramming factors driven by one promoter. While lentivirus has similar rates of reprogramming...control. Additionally, Sendai virus doesn’t rely on promoters for transcriptional regulation, so it requires... mini-plasmids. They contain only a eukaryotic promoter and the cDNA(s) to be expressed and they don’t...phosphatase, expression of embryonic proteins, and promoter demethylation and expression of endogenous pluripotency...

...gRNA to the U6 promoter, and a second plasmid containing the homology arms and an N- or C-terminal tag.... site accepts the sgRNA target sequence for U6 promoter expression and the second site accepts the homology... T2A-TurboGFP-PEST), and small epitope tags (HA, Myc, Strep tag II, AviTag, HaloTag, SpyTag). The Foerstemann...regions of homology to the gene of interest to promote homology directed repair after a CRISPR/Cas9 induced...PCR or by evaluating visual phenotypes. Improving C. elegans fluorescent protein tagging with SapTrap ...called SapTrap to improve CRISPR genomic tagging in C. elegans. SapTrap offers scientists a convenient one...protein tagging systems For those who do not work with C. elegans, several groups have designed modular toolkits...

...to it. The FLAG, hemaglutinin antigen (HA), and c-myc tags have been the workhorses of the affinity tag...step purification with amylose, but wicked huge Myc EQKLISEEDL  1.2 Affinity Frequently used for western...amino acid protein  12 Stability At N-terminus, promotes folding and structural integrity; cleavable. Not... tags, a His tag can be fused to either the N- or C-terminus of a protein. Unlike other epitope tags –...

...sites (thrombin and TEV), and C-terminal tags (StrepII, 3xFLAG, 6xHIS, MBP, Myc). These constructs include...expression plasmids in a high copy backbone with a T7 promoter driving multiple combinations of WT, codon optimized...which drive expression using either TEF1 or GAL1 promoters. These vectors are intended for the high level...

...fluorescent proteins, 8 constitutive promoters, 2 includible promoters, 3 polyA terminators and various pieces...sequences. Several pre-constructed promoter-selection marker or promoter-inducible expression related vectors...all chemically inducible promoters; 3) improving the strength of the promoters used; and 4) optimizing ...ideal promoter/location combination. However, other constructs which contain different promoters and/or...to study the human GM-CSF promoter and enhancer, a finely regulated promoter controlled by a mixture of... Cas9 into n-terminal (Cas9(N)-2xNLS) and c-terminal (Cas9(C)-2xNLS) fragments facilitates target DNA ...activation, the c-terminal cas9 fragment was fused with FK 506 binding protein 12 (Cas9(C)-FKBP-2xNLS) and...

...includes four promoters, six fluorophores with nonoverlapping emission spectra (N- and C-terminal tags...of transcription factors OCT3/4, SOX2, KLF4, and c-MYC is the established model for reprogramming somatic...neurodegenerative diseases, the Cole Lab selected promoters for cell types directly linked to disease, including...article have been deposited with Addgene and include MYC- and FLAG-tagged TZAP constructs (wild-type and truncation... components can be controlled by a variety of promoters for versatile and robust engineering. These components...coselection, analogous to methods previously used in C. elegans, targets the sodium/potassium pump ATP1A1... the B. subtilis Single Gene Deletion Library—Kanamycin and the CRISPRi Essential gene Knockdown library...

...four reprogramming factors- OCT4, SOX2, KLF4, and C-MYC - that induce a differentiated somatic cell to revert...additional pluripotency stage) Less Safety Less (C-MYC, a known oncogene is one of the reprogramming factors...iPSC-derived osteoblasts and osteoclasts together promote bone regeneration in 3D biomaterials. Sci Rep, ...22645348. PubMed Central PMCID: PMC3390875. 25. Patsch, C., et al., Generation of vascular endothelial and smooth... PubMed Central PMCID: PMC4363833. 35. Eguizabal, C., et al., Dedifferentiation, transdifferentiation,...1): p. 82-94. PubMed PMID: 23329641. 36. Jopling, C., S. Boue, and J.C. Izpisua Belmonte, Dedifferentiation...

...(3xFLAG, Myc, V5, or HA). Tag-specific PCR genotyping can tell if HDR completely failed. If C-terminus...Kalantzaki M, Gangoso E, Matjusaitis M, Garcia-Diaz C, Blin C, Grant V, Bulstrode H, Gogolok S et al. 2017. ...will be suitable). For N-terminus tagging, the promoter region of both the alleles should be sequence-...cautiously analyzed for any damage (indels) to the promoter region of the non-HDR allele. For N-terminal tagging...available for human and mouse genomes only and deals with C-terminus epitope tagging. If you work with other model...your crRNAs using other web-based design tools (for C-terminus tagging, pick a crRNA that cuts 8-15 bp downstream...insertion site (within ~8-15 bp from the stop codon for C-terminus tagging) GC content of the crRNA (aim for...

...j.cell.2021.09.018 Choi, J., Chen, W., Suiter, C. C., Lee, C., Chardon, F. M., Yang, W., Leith, A., Daza,...expression ~ ~ ~ ~ ✓   ✓ SV40 and c-Myc nuclear localization sequences Improve translocation..., J., Oyler-Castrillo, P., Ravisankar, P., Ward, C. C., Levesque, S., Jing, Y., Simpson, D., Zhao, A.,...complex, to temporarily inhibit this process and promote copying of the edited strand to the unedited strand... co-express mismatch repair inhibitor MLH1dn Promote desired resolution of heteroduplex     ✓ ...Leukemia Virus (M-MLV) reverse transcriptase to the C-terminus of Cas9 H840A nickase (PE1). But innovations...pegRNAs (Yan et al., 2024). The team fused La to the C-terminal end of PEmax to produce PE7. PE7 significantly...

... 79628 (C) Cv (capsid) N and C 79633 (N) and 79641 (C) NS1 (var: W98G) N and C 79635 NS3 C 79637...79637 NS2B C 79636 NS4A N 79640 NS4B C 79639 NS5 C 79632 (N) and 79631 (C) PrM N and C   Detecting... gRNA from a Drosophila U6:2 promoter and Cas9 from the actin 5C promoter. Addgene’s fly library web page... over 30 promoters and 140 genes, including constructs with unique tissue-specific promoters expressing...tagged BioID2 for N-terminal fusions and Myc tagged BioID2 for C-terminal fusions. Kim, et al. Mol Biol...genomic libraries are cloned downstream of a minimal promoter sequence. Should one of these random stretches...activate its own transcription from the minimal promoter and its strength can be determined from its enrichment...

...targeted gene from its promoter, creating a loss-of-function allele as well as a promoter-FP fusion for the...genome engineering methods for C. elegans were released, signaling a new era in C. elegans research. Homology...of custom genomic modifications, is very robust in C. elegans, and the methods for HDR-mediated modification...the University of North Carolina has made CRISPR in C. elegans even easier - now, one can generate a fluorescent...-based gene tagging approach Multiple features of C. elegans have made CRISPR/Cas9 so successful in this...previously used a transgene selection strategy for their C. elegans/CRISPR experiments, but this strategy required... make it possible fluorescently label proteins in C. elegans, they inserted the SEC into a synthetic intron...

... are repressible promoters regulated? Like inducible promoters, repressible promoters can be regulated...Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region ...natural and engineered repressible promoters. Chemically repressible promoters Tet-Off In its native context... pADH1 displays low promoter activity (~20% activity of the strong yeast promoter pTEF). As ethanol accumulates...that promoter is active. In this way, you can interrogate the activity of uncharacterized promoters. Similarly... QUAS promoter, and the QF transcription factor cannot drive transcription from the UAS promoter. This...repressible promoter systems, but there are certainly more out there! The repressible promoter systems detailed...

...dependent promoters, Baojun Wang’s lab developed a system that can activate sigma-54 dependent promoters (Liu...activation of sigma-54 type promoters. The system was functional in activating promoters in Pseudomonas syringae...that utilizes phage recombination machinery to promote homologous recombination of linear DNA fragments...regulate gene expression were limited to inducible promoters or direct gene knockout. In contrast, CRISPR offers...systems, catalytically dead (dCas9) targeted to a promoter or gene body can repress transcription by physically...and can be more effective targeting either the promoter or the coding sequence and the template or non-template...to multiplex repression. Using a Tet responsive promoter to drive expression of a catalytically dead Cas9...

...hybrid promoter. Although the DE3/T7 RNAP system works well for most experiments, the lac promoter can “...expression  m15 pREP4*, ** Kanamycin (pREP4) Cis-repression of the E. coli T5 promoter (found on vectors such...expression from high-copy number plasmids and powerful promoters will greatly exceed that of any native host protein... accomodate high protein loads using inducible promoter systems (which will be discussed later). In addition...above, many expression plasmids utilize inducible promoters, which are 'inactive' until an inducer such as...the culture to make protein.  The DE3 lysogen/T7 promoter combination is the most popular induction system...available to transcribe the gene of interest from a T7 promoter on the plasmid. Many commercial strains carry ...

...pyogenes (Sp) Cas9 from a Cbh promoter. Cbh is a modified version of CAG promoter that the Zhang lab has tested...imporant is that, upstream of the Cbh promoter, this plasmid has a U6 promoter followed by BbsI restriction sites...site). Finally, in addition to the 2A peptide on its C-terminus, SpCas9 is also fused to a 3X FLAG tag at...SpCas9 is fused to the puromycin resistance gene through a 2A peptide. The puromycin resistance gene enables...causes the ribosome to translate Cas9-2A and the Puromycin resistance gene as separate peptides. PX459 This...predecessor, which had a point mutation in the puromycin resistance gene that made it less effective in...

...from its own promoter. But that doesn’t mean you have to clone and transfect multiple promoter-gRNA constructs...arrays (e.g. for spacers A, B, and C, you could obtain arrays A, B, C, AB, AC, BC, and ABC). Nielsen Lab...can I express more than one gRNA from a single promoter using a plasmid like pX330? Unfortunately, the...of pX330, contains humanized wtCas9 and two U6 promoters. To use this plasmid, you simply order oligonucleotides...contain Type IIS restriction sites flanking the promoter-gRNA construct, but with different sequences adjacent...contain Type IIS restriction sites that flank the promoter-gRNA constructs. When these plasmids are digested...downstream of the 7SK, human U6, mouse U6, or human H1 promoters. If you express fewer than four gRNAs, you’ll ...

...screening of human promoters, the Zhang Lab found a short fragment of human IBA1 promoter (hIBA1a, 466 bp...small RNA-binding exonuclease protection factor) promotes editing (Yan, J et al., 2024). They developed ... expression from ssAAV5s with hIBA1 or hIBA1a promoters in mice. B) Quantifications showing high microglia...a CC-BY-NC-ND license.  Integrating hIBA1a promoter into AAV vectors, either alone or in combination...specificity of GFP expression for the indicated ssAAV5s. C) Quantifications showing high microglia transduction... microglia-targeting AAV plasmids here! Serrano, C., et al. (2023). Simple and Highly Specific Targeting... vectors for CROPseq-multi using selection by Puromycin, Zeocin®, or nuclear expression of BFP2.   ...

...El-Bakkoury, M., Hamacher, T., Cappellaro, C., Vilarino, C., Fleischer, C., Ellerbrok, H., Kamuzinzi, R., Ledent...contains a reporter gene cloned downstream of a promoter containing the target sequence of the DBD. The...as the “prey”) in close enough proximity to the promoter of the reporter gene to activate expression (Figure...organisms, including bacteria, alternative fungi (C. albicans, pC2HB) (7) and mammalian cells. If the ... prey libraries can be screened using both N- and C-terminal fusions of these proteins. This way both ...Two hybrid systems were developed in Saccharomyces cerevisiae in 1989 and are still used extensively ...