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Lenti sgrna ms2 puro backbone

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The Lenti sgRNA (MS2)_puro backbone is a plasmid vector used for the expression of single guide RNA (sgRNA) in lentiviral systems. It contains a puromycin resistance gene for selection of transduced cells. The backbone is designed to express sgRNA with an MS2 stem-loop structure, which can be used for various applications in gene editing and transcriptional regulation.

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Lab products found in correlation

T4 dna ligase buffer, by New England Biolabs (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Lenti ms2 p65 hsf1 hygro, by Addgene (1 mentions) Lentiviral packaging plasmids, by Biosettia (1 mentions) Lenti dcas vp64 blast, by Addgene (1 mentions) Speedy lentivirus purification reagent, by Applied Biological Materials (1 mentions)

Spelling variants of this product

Lenti sgRNA (MS2)_puro optimized backbone (4 citations) Lenti sgRNA (MS2) _puro, (3 citations) Lenti-sgRNA-puro (3 citations)

2 protocols using lenti sgrna ms2 puro backbone

1

Lentiviral sgRNA Vector Construction

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Briefly, the single guide RNA (sgRNA) expression vector lenti sgRNA (MS2)_puro backbone (Addgene, #73795) was digested with BsmBI and was gel-purified. A pair of 20 nt oligos containing the appropriate overhang was then ligated into the vector by mixing 1 μL of the cut vector (normalized to 100-200 ng/mL), 0.5 μL of each primer at a 100 mM stock concentration, 2 μL of 10x T4 DNA ligase buffer, and 1 μL of T4 DNA ligase (NEB #M0202T) into a total ligation volume of 20 μL. Ligations were left at room temperature overnight, and 1 μL of the ligation product was subsequently transformed into 10 μL of Stable 3 component cells. Resulting colonies were verified by Sanger sequencing.
Deng X., Li S., Kong F., Ruan H., Xu X., Zhang X., Wu Z., Zhang L., Xu Y., Yuan H., Peng H., Yang D, & Guan M. (2020). Long noncoding RNA PiHL regulates p53 protein stability through GRWD1/RPL11/MDM2 axis in colorectal cancer. Theranostics, 10(1), 265-280.
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2

Lentiviral Vector Transduction of PCa Cells

Cited 1 time
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Lenti dCAS-VP64_Blast (Addgene plasmid # 61425; http://n2t.net/addgene:61425;
RRID:Addgene_61425), lenti MS2-P65-HSF1_Hygro (Addgene plasmid # 61426;
http://n2t.net/addgene:61426;
RRID:Addgene_61426), and lenti sgRNA(MS2)_puro backbone (Addgene plasmid #
73795; http://n2t.net/addgene:73795;
RRID:Addgene_73795) were a gift from Feng Zhang. Nontargeted and
ARID5A guide RNA (gRNA) sequences used in this study are
shown in Supplemental Table
S2. The top and bottom strand oligonucleotides were annealed, and
inserted into lenti sgRNA(MS2)_puro backbone vector according to the
reference.25 (link)Lentiviral particles were produced in 293T cells transfected with lenti
dCAS-VP64_Blast, lenti MS2-P65-HSF1_Hygro, or lenti sgRNA(MS2)_puro encoding
guide RNAs, and lentiviral packaging plasmids (BioSettia; San Diego, CA) using
Lipofectamine 3000 reagent. The culture supernatants were collected and
concentrated using Speedy Lentivirus Purification reagent (Applied Biological
Materials, Inc.; Richmond, British Columbia, Canada). The viral titer was
determined using 293T cells as described previously.26 PCa cells were transduced for 24 hr at
MOI’s of 1-10 in the presence of 6 μg/mL polybrene.
Ikeuchi W., Wakita Y., Zhang G., Li C., Itakura K, & Yamakawa T. (2021). AT-rich interaction domain 5A regulates the transcription of interleukin-6 gene in prostate cancer cells. The Prostate.
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