... BL 2012. Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression...delay the closing of the channel 470(activ.), 590(inact.) ReaChR Red-activatable variant of CrChR2 590 Channelrhodopsins...chloride pump from Haloarcula (Halobacterium) salinarum (strain Shark), used to silence neuronal activity...short or long periods of neuronal activation/inactivation periods. These can range from msec (hChR2) to...expression of the opsin and the area that is being illuminated. There are several different ways to control ...especially powerful when combined with site specific recombinase technology like Cre-lox, which allows you to ...one half of a given protein, such as the Cre recombinase, is fused to Cry2 and the other half to CIB1,...

... Rep and Cap, flanked by two 145 base inverted terminal repeats (ITRs). These ITRs base pair to allow ...is necessary for lytic cycle activation. When recombinant AAV (rAAV) is used for research purposes, the...the Rep protein is supplied in trans , eliminating the ability of rAAV to integrate into its preferred ...AAV8 Photoreceptor Cells AAV2, AAV5, AAV8 RPE (Retinal Pigment Epithelium) AAV1, AAV2, AAV4, AAV5, AAV8...spontaneously annealing, upon infection, which eliminates the requirement for host cell DNA synthesis. ...increasing packaging capacity depends on homologous recombination. In this method, a gene is divided between two...sequence overlap. Co-expression induces homologous recombination and expression of the full-length transgene ...

...T7 promoter, forward primer T7 Terminal GCTAGTTATTGCTCAGCGG T7 terminator, reverse primer Tac promoter ...GCAAATGGCATTCTGACATCC (Invitrogen) For Pichia vectors with AOX1 terminator, reverse primer 5'AOX1 GACTGGTTCCAATTGACAAGC ...TAGAAGGCACAGTCGAGG (Invitrogen) Bovine growth hormone terminator, reverse primer Bglob-intron-F CTGGTCATCATCCTGCCTTT...primer CRE-R GCAAACGGACAGAAGCATTT 5' end of Cre recombinase, reverse primer CYC1 GCGTGAATGTAAGCGTGAC (Invitrogen...Invitrogen) CYC1 transcription termination signal, reverse primer DsRed1-C AGCTGGACATCACCTCCCACAACG (BD... GTGGTTTGTCCAAACTCATC (Invitrogen) SV40 polyA terminator, reverse primer Ecdysone Forward CTCTGAATACTTTCAACAAGTTAC...hGH-PA-R CCAGCTTGGTTCCCAATAGA Human growth hormone terminator, reverse primer hrGFP-R TCCCCGAGTACCACTTCATC ...

...interest: The transgene sequence is flanked by long terminal repeat (LTR) sequences, which facilitate integration...line that stably expresses Gag-Pol and/or Env, eliminating the need to deliver these genes in trans by specific...feature may be useful for the transduction of terminally differentiated (non-dividing) cells. Both lentiviruses... only in lentivirus. Are γ-retroviruses self-inactivating (SIN)? They can be. Plasmids that encode SIN...export. LTR Subcomponents: U3 R U5 in cis LTR; Long terminal repeats; U3-R-U5 regions found on either side ...U5 and the rest of the provirus. 3' LTR in cis Terminates transcription started by 5' LTR by the addition...

...transcription factors bind. Distal Promoter The final portion of the promoter region is called the distal...Constitutive Mammalian promoter from phospholycerate kinase gene TRE Inducible Tetracycline response element...Plasmids 101: Repressible Promoters Plasmids 101: Terminators and PolyA signals...

...in mammalian cells. sin 3’LTR 3’ Self-inactivating long terminal repeat. f1 ori f1 bacterial origin of... Map of pLKO.1 containing an shRNA insert. The original pLKO.1-TRC cloning vector has a 1.9kb stuffer ...the transduced cells. hPGK Human phosphoglycerate kinase promoter drives expression of puromycin. Puro R...bacterial origin of replication. 5’LTR 5’ long terminal repeat. RRE Rev response element. A.3 Related ...repeats, especially repeated Ts because polyT is a termination signal for RNA polymerase III. Note that these...and ligated into the pLKO.1 vector, producing a final plasmid that expresses the shRNA of interest. C.1...buffer 1 1 μL AgeI add ddH 2 O to bring to 50 μL final volume Incubate at 37°C for 2 hours. Purify with...

...protocol is composed of steps that separate out contaminants from an impure AAV preparation. The 15% iodixanol...macromolecules. The 40% and 25% steps are used to remove contaminants with lower densities, including empty capsids...mg of MgCl 2 and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL. Sterilize by passing through a... of MgCl 2 , and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL. Sterilize by passing through a....5 mL to 1 mL fractions per tube. Avoid the proteinaceous material near the 40–25% interface. Pro-Tip ...per tube, taking care to avoid collecting the proteinaceous material at the 40–25% interface. Repeat for.... Image adapted from Zolotukhin, S., et al. "Recombinant adeno-associated virus purification using novel...

...mL pipette 5–10 times. The final volume in the well is 1.5 mL, and the final Polybrene concentration is...high glucose, pyruvate, Corning, 10-013-CV Heat-inactivated FBS L-alanyl-L-glutamine (or alternative stable...500 mL DMEM, high glucose, pyruvate 55 mL heat inactivated premium grade, fetal bovine serum 5 mL glutaGRO...new aspirating pipette for each well as to not contaminate cell conditions with others. Gently add 1.5 mL...master mix for 9 samples. Component Volume 9X Volume Final Concentrations 2X ddPCR Supermix for Probes, no ...from the appropriate dilutions to calculate the final infectious titer. Tips and Troubleshooting We recommend...freezer blocks during use. To reduce the risk of contaminating reagents we recommend making small aliquots ...

...method for decontaminating must be used for proper disposal. Liquid BSL-2 waste can be decontaminated in a ...biohazardous waste. Dispose of waste that may be contaminated with pathogens or chemicals that present a danger...biohazard containers. Biohazardous materials are decontaminated prior to disposal, commonly done with an autoclave...underneath a hood. Clean any spills immediately, and decontaminate as necessary. For large spills that cannot be...participating in BSL-2 work receives a hepatitis B vaccination or titer prior to starting work in the laboratory...a final concentration of 10% bleach for 30 minutes before pouring down the drain. Solid BSL-2 waste can...

...Bottlenecks, fitness differences, and plasmid recombination can all impact the representation of individual...libraries may require modifications dictated by the originating laboratory for optimal results. If you obtained...replication and antibiotic selection). This recombination, at a low rate, is not typically a problem as...lentiviral particles. See below for options if the recombinant band makes up a significant proportion of the...electrocompetent cells that are suitable for unstable or recombination-prone DNA. The use of electrocompetent cells...step. I got a heavy recombined band by digest. Recombination is thought to be in part related to selective...burden on the bacteria. Bacteria that contain a recombinant plasmid likely grow faster in the closed system...

...FBS can be purchased already heat-inactivated, or it can be inactivated in the lab by heating to 56 °C for...transiently-transfected cells, increases reproducibility, as it eliminates the variation associated with repeated transient...antibiotic selection on transduced cells enables elimination of untransduced cells, resulting in a more homogenous...glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore TR-1003-G...bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5mL of 100X glutaGRO. Store at 4 °C. ...made with DMEM complete + 10 µg/mL polybrene, the final concentration of polybrene in each well should be...

...purified AAV samples with DNase I to eliminate any contaminating plasmid DNA carried over from the production... a blend of dTTP/dUTP to minimize carryover contamination. The master mix should also contain an internal...help stabilize the standards add carrier DNA to a final concentration of 4 ug/mL to each standard dilution...at least 10 times, and use at least half of the final volume (mix with >50 uL if your well contains 100...adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum Gene Ther Methods. 2012 ...

...In our case, we will add TAAGCA, resulting in a final Forward Primer sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC...amplification. We can start similarly, taking the final 18bases of the ORF, including the stop codon (5'...TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a final Reverse Primer sequence of 5’-TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA... higher competency cells. Additionally, if your final product is going to be very large (>10kb) you might...plasmid to insert. Isolate the Finished Plasmid Finally, you will need to pick individual bacterial colonies... you use, it is important that you sequence the final product. Reference Page Top Index...

...FBS can be purchased already heat-inactivated, or it can be inactivated in the lab by heating to 56 °C for... 2 Cellstack 2, Corning 3269, 1272 cm 2 Heat-inactivated FBS (HI-FBS) Pro-Tip Different brands and lots...bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of glutaGRO 11 mL of 200 mM L-alanyl-L-glutamine... g of NaCl into deionized water and adjust to a final volume of 1000 mL. Stir at RT until fully dissolved...0.45 μm PES membrane. Add 40% PEG such that the final PEG concentration is 8%. Add 25 mL of PEG solution...

...FBS can be purchased already heat inactivated or it can be inactivated in the lab by heating to 56°C for...chloroquine Polyethylenimine, linear MW 25,000 Da Heat-inactivated FBS 1x PBS pH 7.4 without calcium or magnesium...bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of glutaGRO. Store at 4 °C. Pro-Tip...of 25 mM chloroquine to the conical so that the final concentration in the dish (once transfection mixes...

...FBS can be purchased already heat-inactivated, or it can be inactivated in the lab by heating to 56 °C for...glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore TR-1003-G...bottle of DMEM high glucose, add 55 mL of heat inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C.... Plate (μL) Volume of Cells Added to Plate (μL) Final Viral Dilution 1:10 100 of Stock Virus 900 150 1,350...

...prevent contamination. When not in use, the tip box should be closed to prevent contamination. Reading...for measuring different samples without cross contamination as long as the tip is changed between samples...not touch the tips with your fingers to avoid contaminating the tip or puncturing your glove. Ensure that...pipette tip into the container in order to avoid contaminating the contents. Once the end of the tip is submerged...

...RNAse to the supernatant after step 6 to eliminate RNA contamination. This is included in the resuspension...genomic DNA will become sheared and will therefore contaminate your purified plasmid DNA. Incubate solution ...Phenol-chloroform extraction removes remaining contaminant proteins and RNase A from the DNA sample. When... This will help to remove proteins and other contaminants from the plasmid DNA. It is also advisable to...

...glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS 1X PBS pH 7.4 without calcium or magnesium...bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C.... integrated transgene could be lost through recombination. Some researchers choose to maintain cells in...times that volume, which is 12.5 µL. To make the final 5 cell/mL solution, transfer 12.5 µL of the 1:100...larger culture dishes, being sure not to cross contaminate the independent monoclonal lines. Pro-Tip When...

...droplet digital PCR (ddPCR) to titer purified recombinant Adeno-associated viral vectors (AAV). This protocol...window) . Before Starting To reduce the risk of contaminating reagents we recommend making small aliquots ...generation (droplet generation BSC) to ensure no contamination of the PCR reagents and the NTC with virus. ... for 9 samples. ITR Master Mix Volume 9X Volume Final Concentration 2X ddPCR Supermix for Probes, no dUTP... is prepared for each AAV sample, and the three final dilutions are assayed. The samples that are assayed...