lenti MS2-P65-HSF1_Hygro

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.	There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.
Protocol tips
There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

Molecular Clones.
The previously described lentiviral vectors encoding the MS2-p65-HSF1 (61426) and dCAS-VP64 (61425) fusion proteins and the sgRNA(MS2) (61427) chimeric RNA (12) were obtained from Addgene (courtesy of Feng Zhang, Broad Institute, Cambridge, MA). The gene inserts from MS2-p65-HSF and dCAS-VP64 were amplified by PCR and cloned into pcDNA3 to create pMS2-p65-HSF1, and pdCAS-VP64. Oligonucleotides encoding sgRNAs, designed to be specific for either the A3B or A3G promoter regions (29, 30), were ligated into sgRNA(MS2). The U6 promoter cassettes from sgRNA(MS2), with either no sgRNA or containing the inserted promoter-specific sgRNA, were amplified by PCR and then cloned into pGEM3Zf(+) as EcoRI/HindIII fragments to create pGU6, pGsgB1, pGsgB2, pGsgG1, and pGsgG2.

The lentiviral dual A3G guide expression vector was generated by modifying MS2-p65-HSF1 (Addgene 61426) as follows: the A3G sgG2 guide cassette from pGsgG2 was amplified by PCR and inserted into the unique NheI site in MS2-p65-HSF1 to create MS2-p65-HSF1/sgG2. The A3G sgG1 guide cassette from pGsgG1 was also amplified by PCR and inserted into the unique EcoRI site in MS2-p65-HSF1/sgG2 to create the dual guide lentiviral expression vector MS2-p65-HSF1/sgG(1+2).

The FLuc-based indicator construct pGL4.10 (Promega) was digested with BglII and HindIII and complementary synthetic oligonucleotides containing a minimal TATA box were annealed and inserted. This modified pGL4.10+TATA was then digested with XhoI and BglII and oligonucleotides were annealed and inserted to generate the indicator constructs pFLucA3Brep1, pFLucA3Brep2, pFLucA3Brep3, pFLucA3Grep1, pFLucA3Grep2, and pFLucA3Grep3. Oligonucleotide sequences used to generate the sgRNA expression constructs and pFLuc reporter plasmids are listed in SI Materials and Methods.

The HIV-1 proviral FLuc expression vectors pNL-HXB-FLuc (HIV-1WT) and pNL-HXB-FLucΔVif (HIV-1ΔVif), as well as the VSV glycoprotein expression plasmid pHIT/G, have been previously described (13, 14). The gene for NLuc (Promega) was amplified by PCR and inserted between the unique NotI and XhoI sites of pNL-HXB-LucΔVif (HIV-1ΔVif) to generate pHIV-1-NLuc-ΔVif. The identical strategy was used to construct pHIV-1-NLucWT.

Cell Culture and Transfection.
HeLa and 293T cells were cultured in DMEM supplemented with 5% (vol/vol) FBS and gentamicin (Life Technologies). The human CD4+ T-cell line CEM-SS (776, NIH AIDS Reagent Program) was maintained in RPMI-1640 medium, supplemented with 10% (vol/vol) FBS and gentamicin. All HeLa and 293T transfections were performed using polyethylenimine (PEI). All CEM-SS transfections were performed using Lipofectamine LTX (ThermoFisher).

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Manufacturer protocol

Download the product protocol from Addgene for lenti MS2-P65-HSF1_Hygro below.

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