pMXs-IRES-Bsd Retroviral Expression Vector

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	For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

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
For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Publication protocol

Vector construction
The GFP and FLAG-tagged C16orf80, C3orf17, and C9orf114 expression vectors were
constructed by cloning, via Gibson assembly, cDNA inserts generated by PCR from a KBM7
cDNA library into versions of the pMXs-IRES-Bsd vector containing a C-terminal GFP or
FLAG tag. For sgDDX3Y rescue experiments, GFP and DDX3X expression vectors were
constructed by cloning, via Gibson assembly, cDNA inserts generated by PCR from a GFPtagged pMXs-IRES-Bsd vector (for GFP) and a KBM7 cDNA library into the pMXs-IRES-Bsd
vector (for DDX3X). For live-cell imaging, the EGFP cassette in pEGFP-C1-Fibrillarin was
replaced with turboRFP.
Genome-wide lentiviral sgRNA library construction
Oligonucleotides were synthesized on the CustomArray 90K arrays (CustomArray Inc.) as two
separate sub-pools. PCR was performed to incorporate overhangs compatible for Gibson
Assembly (NEB) into lentiCRISPR-v1 linearized with BsmBI (primer sequences provided
below). Gibson Assembly reaction products were transformed into E. cloni 10G SUPREME
electrocompetent cells (Lucigen). To preserve the diversity of the library, at least 20-fold
coverage in library representation was recovered in each transformation and grown in liquid
culture for 16-18 hours.
PCR primers for library amplification
F-GGCTTTATATATCTTGTGGAAAGGACGAAACACCG
R-CTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC
Virus production and transduction
Virus was produced by co-transfecting the transfer vector of interest with VSV-G envelope
plasmid and Delta-Vpr (lentiviral) or Gag-Pol (retroviral) packaging plasmids into HEK-293T
cells using XTremeGene 9 transfection reagent (Roche). Media was changed 24 hours after
transfection and the virus-containing supernatant was collected 72 hours after transfection and
passed through a 0.45 μm filter to eliminate cells. Target cells in 6-well tissue culture plates were
infected in media containing 8 μg/mL of polybrene by centrifugation at 2,220 RPM for 45 minutes. 24 hours after infection, virus was removed and cells were selected with the appropriate
antibiotics.

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Manufacturer protocol

Download the product protocol from Cell Biolabs for pMXs-IRES-Bsd Retroviral Expression Vector below.

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