...D., & Kay, M. A. (2003). From virus evolution to vector revolution: use of naturally occurring serotypes...engineering and evolution of adeno-associated viral vectors for enhanced gene transfer . Pharmaceutical Research...enhanced function after intravenous administration, distributing widely throughout the organism to target large...viral preps . AAV retrograde (AAVrg) Directed evolution has also been employed to engineer an rAAV serotype...capsids, and capsids created through directed evolution. Created with BioRender.com. Common Uses of AAV...office can provide more information on your institution's best practices with regard to AAV-related research...vector integration . Current Opinion in Molecular Therapeutics, 11 (4), 442–447. PMID: 19649989 (Link opens...

...close enough to cause a DSB. The nickase system is utilized in many CRISPR systems to achieve specific genetic... in nature, but have been created by directed evolution of the Escherichia coli TadA, a tRNA adenosine...window. These systems are well-suited to directed evolution applications. Examples of these base editing systems...activity. Knockout libraries often target 5′ constitutively expressed exons, while activation and repression...repression, many CRISPR epigenetic tools require constitutive expression of the dCas9 fusion and/or expression...CAPTURE system, developed by the Jian Xu lab , utilizes biotin tagging of dCas9 by fusing a biotin acceptor... Kim, D., Kim, S., & Kim, J. (2018). Directed evolution of CRISPR-Cas9 to increase its specificity. Nature...

... factor 1 alpha CAG Constitutive Strong hybrid mammalian promoter PGK Constitutive Mammalian promoter ...Description T7 Constitutive but requires T7 RNA polymerase Promoter from T7 bacteriophage Sp6 Constitutive but requires...Research Promoter Expression Description CMV Constitutive Strong mammalian promoter from human cytomegalovirus...Inducible Tetracycline response element promoter U6 Constitutive Human U6 nuclear promoter for small RNA expression...polymerase Promoter from Sp6 bacteriophage lac Constitutive in the absense of lac repressor (lacI or lacIq...

...recombine DNA? There are many methods that have been utilized over the years to move around pieces of DNA. Oftentimes...in the DNA. In restriction cloning, scientists utilize specific restriction enzymes to cut dsDNA of interest...reducing the amount of time required for cloning. To utilize this approach, the fragment of DNA that you would...pairs adenine (A) and thymine (T). TOPO® cloning utilizes the Taq polymerase which naturally leaves a single...traditional restriction enzyme cloning. Scientists have utilized this approach to create compatible custom overhangs... 100kb) compared to other cloning methods that utilize bacteria where it becomes progressively more difficult...

...for light-induced cAMP modulation. Utilizes BLUF (blue light utilizing FAD) domains. 441 Phytochromes BphP1...technologies developed for optogenetics research utilize light to detect, measure, and control molecular...expressed under the control of the vesicular gamma aminobutyric acid (GABA) transporter (VGAT) and ChR2 is thus...figure above, this light induced binding can be utilized to control the localization of a protein of interest...of neural activity, with single-spike temporal resolution. PLoS One. 2(3):e299. PMID 17375185 Kim B, Lin...

...followed by transduction is necessary to properly utilize lentiviral vectors. Some lentiviral plasmids (mostly...is still random and difficult to predict. In therapeutic applications, like chimeric antigen receptor ...particles. However, VSV-G is toxic under long-term, constitutive expression. In order to make stable cell lines...office can provide more information on your institution's best practices with regard to lentiviral-related...Third-generation plasmids use a hybrid 5' LTR with a constitutive promoter such as CMV or RSV. 3' LTR in cis Terminates...

...vivo and the receptors exhibited high levels of constitutive activity, making them less than ideal. DREADDs...insensitivity to endogenous ligands, have low constitutive activity, and their activating ligands have ...This DREADD was shown to have a small amount of constitutive activity, and is not widely used. There is also...cross the blood-brain barrier in rats, and the utility of DREADDs in experiments in this species may be...

...particles. However, VSV-G is toxic under long-term, constitutive expression. In order to make stable cell lines...and provide stable integration of the intended therapeutic. While similar to lentiviral vectors, they offer...office can provide more information on your institution's best practices with regard to gamma-retroviral-related...Some newer plasmids use a hybrid 5' LTR with a constitutive promoter such as CMV or RSV. 3' LTR in cis Terminates...

... DNA sequencing of the plasmid. Primers can be utilized to verify the sequence of the insert or other ...ampicillin. Preparing Antibiotics Create a stock solution of your antibiotic. Unless otherwise indicated...dH 2 0. Addgene recommends making 1000X stock solutions and storing aliquots at -20°C. To use, dilute ..., like Sanger, is based on DNA replication and utilizes modified fluorescently tagged nucleotides. During...

...response quickly, which has limited their use in therapeutic applications. However, this has also made them...office can provide more information on your institution's best practices with regard to AdV research. ...for outbreak pathogens . Human Vaccines & Immunotherapeutics, 13 (12), 3020–3032. https://doi.org/10.1080...

...expression vector Cas enzyme promoter can be constitutive (CMV, EF1alpha, CBh) or inducible (Tet-ON); ...genetic knockouts, gRNAs commonly target 5′ constitutively expressed exons, which reduces the chances ...

...technologies developed for optogenetics research utilize light to detect, measure, and control molecular...

...bind to a protein is powerful, but in order to utilize it in the lab, researchers must be able to detect...