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Pltr g

Manufactured by Addgene
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Sourced in United Kingdom

The PLTR-G is a lab equipment product. It serves as a core function, but a detailed description while maintaining an unbiased and factual approach is not available.

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Lab products found in correlation

Pcd nl bh ddd, by Addgene (3 mentions) 0.45 μm sterile filter, by Merck Group (1 mentions) Plko 1 puro plasmid, by Addgene (1 mentions) Polyfect transfection reagent, by Qiagen (1 mentions) Pcmv dr8.2 dvpr, by Addgene (1 mentions) Midiprep plasmid isolation kit, by Qiagen (1 mentions) Lenticrispr v2 vector, by Addgene (1 mentions) Lenticrispr v2, by Addgene (1 mentions) Sigma mission library, by Merck Group (1 mentions) Polybrene, by Merck Group (1 mentions) Puromycin, by Merck Group (1 mentions)

4 protocols using pltr g

1

Lentiviral Particle Production and Titration

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Lentiviral expression plasmids were transfected, along with the envelope plasmid (pLTR-G, Addgene #17532) and the packaging plasmid (pCD/NL-BH*DDD, Addgene #17531), into 293T cells, using polyethylenimine, and the virus-containing culture media were harvested at 48 and 72 h. The culture media were centrifuged (500 g, 10 min), filtered through a 0.2 μm syringe filter (6780-2502, Puradisc, Whatman, Maidstone, United Kingdom), ultracentrifuged twice (50,000 g, 2 h each), and finally concentrated by 400-fold and resuspended in DPBS. The viral titer was determined using 293T cells.
Mishima T., Sadovsky E., Gegick M.E, & Sadovsky Y. (2016). Determinants of effective lentivirus-driven microRNA expression in vivo. Scientific Reports, 6, 33345.
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2

Lentiviral Transduction of shRNA in HEK293T Cells

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HEK293T cells
were seeded in a 10 cm dish at 30% confluence 1 day prior to transfection
with 4 μg of pLKO.1 puro plasmid (Addgene no. 8453) for shRNA
expression, 1 μg of pLTR-G (Addgene no. 17532) envelope plasmid,
and 3 μg of pCMV-dR8.2 dvpr (Addgene no. 8455) package plasmid
together with 40 μL of PolyFect transfection reagent (Qiagen).
The medium was replaced 12 h after transfection. After 48 h, viral
particles were harvested from the culture medium and filtered with
a 0.45-μm sterile filter (Millipore). Cells were transduced
with a lentivirus for 48 h and subsequently screened with 1.0 μg/mL
puromycin. The shRNA sequences are listed in Table S4.
He X., Yuan J., Gao Z, & Wang Y. (2023). Promoter R-Loops Recruit U2AF1 to Modulate Its Phase Separation and RNA Splicing. Journal of the American Chemical Society, 145(39), 21646-21660.
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3

Generating CRISPR-Cas9 Knockout Cell Lines

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A stable knockout of ADD1 or ADD3 in H1573 and 16HBE14o cells was carried out using a CRISPR-Cas9 technology. The guide oligonucleotide sequences used for knocking out ADD1 and ADD3 are shown in the Supplemental Table 1. The guide oligonucleotides were phosphorylated, annealed and cloned into the BsmBI site of a lentiCRISPR v2 vector (Addgene, 52961) according to a published protocol [43 (link), 44 (link)], Obtained constructs were verified with sequencing. Transfer plasmids possessing annealed guide oligonucleotides were transformed into recombination-deficient Stbl3 bacteria and amplified plasmids were isolated from the bacteria using Qiagen midi prep plasmid isolation kit. Lentiviruses were produced by transfecting HEK-293T cells with the transfer lentiCRISPR v2 plasmids and packaging plasmids pLTR-G (Addgene, 17532) and pCD/NL-BH*DDD (Addgene, 17531). Viral supernatants were collected 48 and 72 h after transfection and used to infect H1573 and 16HBE14o cells. After 24 h of the infection, the lentivirus-containing medium were replaced with fresh cell culture medium containing puromycin (2 μg/ml for H1573 cells and 10 μg/ml forl6HBE14o cells) and puromycin-resistant cells were collected after 7 day selection.
Lechuga S., Amin P.H., Wolen A.R, & Ivanov A.I. (2018). Adducins inhibit lung cancer cell migration through mechanisms involving regulation of cell-matrix adhesion and cadherin-11 expression. Biochimica et biophysica acta. Molecular cell research, 1866(3), 395-408.
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4

Lentiviral shRNA for Htra1 Knockdown

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Lentiviral shRNA constructs specific for Htra1 were purchased from the Sigma Mission library (Sigma-Aldrich) and consisted of TRCN0000031484 (shHtra184) and TRCN0000031486 (shHtra186). The SHC002 non-target control shRNA construct (shControl) was kindly provided by Prof. Michael Ehrmann (University of Duisburg-Essen, Germany). All shRNA construct were cloned into the pLKO.1-puro vector. In order to generate shRNA-expressing lentiviral particles, HEK293T cells were transfected with shRNA plasmids, in combination with packaging plasmid pCD/NL-BH*DDD (Addgene plasmid #17531) [36 (link)] and envelope plasmid pLTR-G (Addgene #17532) [37 (link)] using calcium phosphate co-precipitation, and lentiviral particles collected at 24 and 48 h. C3H10T1/2 cell cultures were transduced with virus, together with 8 μg/ml polybrene (Sigma-Aldrich), and medium refreshed with normal growth medium after 24 h. Transduced cells were selected for 1 week in the presence of 2 μg/mL puromycin (Sigma Aldrich), and subsequently seeded at 7’000 cells/cm2 in cell culture plates.
Filliat G., Mirsaidi A., Tiaden A.N., Kuhn G.A., Weber F.E., Oka C, & Richards P.J. (2017). Role of HTRA1 in bone formation and regeneration: In vitro and in vivo evaluation. PLoS ONE, 12(7), e0181600.
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