Short hairpin RNAs (shRNAs) against mouse Ubap2 (shUbap2_#1: TRCN0000241149 and shUbap2_#2: TRCN0000241151), mouse Cdh1 (shCdh1_#1: TRCN0000042578, shCdh1_#2: TRCN0000042579, shCdh1_#3: TRCN0000042580, shCdh1_#4: TRCN0000042581, and shCdh1_#5: TRCN0000042582), and mouse Fosl1 (shFosl1_#1: TRCN0000042684, shFosl1_#2: TRCN0000042685, shFosl1_#3: TRCN0000042687, shFosl1_#4: TRCN0000310960, and shFosl1_#5: TRCN0000316021) cloned into the pLKO.1-puro vector were purchased from Sigma-Aldrich (Supplementary Table 7 ). For Ubap2 overexpression, mouse Ubap2 cDNA was amplified from mouse testis cDNA library using PCR and cloned into the pDON-5 Neo vector (Takara Bio Inc.). shRNAs for Ubap2, Cdh1, and Fosl1 and Ubap2 overexpression constructs were transfected using pPACKH1TM Lentivector Packaging kit (System Biosciences) into 293TN cells for 72 h using Lipofectamine 3000 (Thermo Fisher Scientific Inc.) and then the collected supernatants were used to treat MC3T3-E1 cells or primary monocytes. pLKO.1-puro empty vector and pDON-5 Neo empty vector were used as negative controls.
Pdon 5 neo vector
Manufactured by Takara Bio
Sourced in Japan
The PDON-5 Neo vector is a plasmid designed for gene expression in eukaryotic cells. It contains a neomycin resistance gene, allowing for selection of cells that have successfully incorporated the vector. The vector also includes a multiple cloning site for insertion of genes of interest.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Plko 1 puro vector, by Merck Group (1 mentions)
Ppackh1tm lentivector packaging kit, by System Biosciences (1 mentions)
Dulbecco modified eagle medium (dmem), by Promega (1 mentions)
Ht1080, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
H1299, by American Type Culture Collection (1 mentions)
Ht1080, by American Type Culture Collection (1 mentions)
B16 f1 melanoma, by American Type Culture Collection (1 mentions)
Pureyield plasmid miniprep system, by Promega (1 mentions)
Noti hf, by New England Biolabs (1 mentions)
Spectrum compact ce system, by Promega (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
3 protocols using pdon 5 neo vector
1
Genetic Manipulation of Ubap2, Cdh1, and Fosl1 in Cells
2
APN/CD13 Overexpression in B16-F1 Melanoma
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HT1080, B16-F1 melanoma, H1299 and A549 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). PC14 cells were obtained from Immuno-Biological Laboratories (Gunma, Japan). HT1080, B16-F1, and A549 cells were cultured in DMEM (Gibco, Grand Island, NY, USA), and H1299 and PC14 cells were cultured in RPMI 1640 Medium (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin. All cells were incubated at 37°C in an atmosphere containing 5% CO2.
A retroviral vector (pDON-5neoCD13; Takara Bio, Shiga, Japan) containing APN/CD13 cDNA and a control pDON-5neo vector were transfected into B16-F1 melanoma cells according to the manufacturer’s protocol. Transfectants resistant to 1 mg/mL G-418 (Promega, Madison, WI, USA) were selected and maintained in DMEM with 10% FBS and 1 mg/mL G-418. From these B16-F1 transfectants, one cell line that expressed high levels of APN/CD13 (APN-B16 cells) and another cell line that did not express APN/CD13 (control-B16 cells) were selected. The expression levels of APN/CD13 were determined by flow cytometry and real-time PCR.
A retroviral vector (pDON-5neoCD13; Takara Bio, Shiga, Japan) containing APN/CD13 cDNA and a control pDON-5neo vector were transfected into B16-F1 melanoma cells according to the manufacturer’s protocol. Transfectants resistant to 1 mg/mL G-418 (Promega, Madison, WI, USA) were selected and maintained in DMEM with 10% FBS and 1 mg/mL G-418. From these B16-F1 transfectants, one cell line that expressed high levels of APN/CD13 (APN-B16 cells) and another cell line that did not express APN/CD13 (control-B16 cells) were selected. The expression levels of APN/CD13 were determined by flow cytometry and real-time PCR.
3
Retroviral Vector Expressing Pig TMPRSS2
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To generate a retroviral vector expressing pig TMPRSS2, the coding sequence of pig TMPRSS2 was synthesized according to the amino acid sequences deposited in GenBank (Acc. Num: NP_001373060) with codon optimization to pig cells (Integrated DNA Technologies, Inc., Coralville, IA, USA). The synthesized DNA sequence is summarized in the S1 File . Synthesized DNA was cloned into the pDON-5 Neo-vector (TaKaRa, Kusatsu, Japan, Cat# 3657), which was prelinearized with NotI-HF (New England Biolabs (NEB), Ipswich, MA, USA, Cat# R3189L) and BamHI-HF (NEB, Cat# R3136L) using an In-Fusion HD Cloning Kit (TaKaRa, Cat# Z9633N). Plasmids were amplified using NEB 5-alpha F′ Iq competent Escherichia coli (NEB, Cat# C2992H) and extracted using the PureYield Plasmid Miniprep System (Promega, Madison, WI, USA, Cat# A1222). The plasmid sequence was verified using a SupreDye v3.1 Cycle Sequencing Kit (M&S TechnoSystems, Osaka, Japan, Cat# 063001) with a Spectrum Compact CE System (Promega).
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