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Blasticidin s hydrochloride

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Blasticidin S hydrochloride is a broad-spectrum antibiotic used as a selection marker in cell culture and molecular biology applications. It inhibits protein synthesis by binding to the 60S ribosomal subunit. Blasticidin S hydrochloride is commonly used to select for successfully transfected or transduced cells that express a blasticidin resistance gene.

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Lab products found in correlation

Lipofectamine ltx, by Thermo Fisher Scientific (3 mentions) Plus reagent, by Thermo Fisher Scientific (3 mentions) Ham s f 12 nutrient mixture, by Thermo Fisher Scientific (2 mentions) Megm bullet kit, by Lonza (1 mentions) Mammary epithelial growth medium, by Lonza (1 mentions) Lipofectamine ltx and plus reagent, by Thermo Fisher Scientific (1 mentions) Chicken serum, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions) Fetal bovine serum (fbs), by Biowest (1 mentions)

6 protocols using blasticidin s hydrochloride

1

Engineered Chromosomes in Cell Lines

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Hypoxanthine phosphoribosyltransferase (HPRT)-deficient Chinese hamster ovary (CHO) cells (JCRB0218) carrying the alphoidtetO-HAC were maintained in Ham's F-12 nutrient mixture (Invitrogen, USA) plus 10% fetal bovine serum (FBS) with 8 μg/ml Blasticidin S Hydrochloride (Funakoshi, Japan). After insertion of a TAR/BAC/BRCA1 construct and the eGFP gene into alphoidtetO-HAC, CHO cells retaining the tetO-eGFP-BRCA1-HAC were maintained in HAT-containing medium. The BRCA1-deficient human ovarian cancer cell line UWB1.289 carrying a germline BRCA1 mutation within exon 11 and having a deletion of the wild-type allele (27 (link)) was obtained from the American Type Culture Collection (ATCC; Manassas, USA). These cells were maintained in 50% RPMI1640 medium containing 50% MEGM (Mammary Epithelial Growth Medium from Clonetics/Lonza) (MEGM Bullet Kit; CC3150) made of Mammary Epithelial Basal Medium (MEBM) basal medium and SingleQuot plus 3% FBS. Swine testis (ST) cells (ATCCRCRL-1746) were obtained from the ATCC. The base medium for ATCCRCRL-1746 cells is ATCC-formulated Eagle's Minimum Essential Medium (Catalog No. 30–2003) plus 10% of FBS.
Kononenko A.V., Bansal R., Lee N.C., Grimes B.R., Masumoto H., Earnshaw W.C., Larionov V, & Kouprina N. (2014). A portable BRCA1-HAC (human artificial chromosome) module for analysis of BRCA1 tumor suppressor function. Nucleic Acids Research, 42(21), e164.
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2

CRISPR-Cas9 Genome Editing in Cell Lines

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HeLa, HCS-2, and SKG-I cell lines were transfected with pCMV-Cas9-HA-IRES-bsr using Lipofectamine LTX and Plus Reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Transfected cells were selected by culturing in cell culture media containing 10 µg/ml of Blasticidin S Hydrochloride (Funakoshi, Tokyo, Japan) to collect single colonies.
Yoshiba T., Saga Y., Urabe M., Uchibori R., Matsubara S., Fujiwara H, & Mizukami H. (2018). CRISPR/Cas9-mediated cervical cancer treatment targeting human papillomavirus E6. Oncology Letters, 17(2), 2197-2206.
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3

SKOV-3 and SHIN-3 Co-Transfection

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The co‐transfection of SKOV‐3 and SHIN‐3 cells with pCMV‐Cas9‐HA‐IRES‐bsr and pRGEN‐U6‐sgRNA or pRGEN‐U6‐sgVASH2 was conducted using Lipofectamine LTX and Plus Reagent (Thermo Fisher Scientific, Inc.). After culturing on cell culture media supplemented with 10 μg/ml of blasticidin S hydrochloride (Funakoshi, Tokyo, Japan) to obtain single colonies, transfected cells were selected.
Koyanagi T., Saga Y., Takahashi Y., Tamura K., Yoshiba T., Takahashi S., Taneichi A., Takei Y., Urabe M., Mizukami H, & Fujiwara H. (2021). Knockout of vasohibin‐2 reduces tubulin carboxypeptidase activity and increases paclitaxel sensitivity in ovarian cancer. Cancer Medicine, 10(8), 2732-2739.
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4

sFlt-1 and VASH1 Expression Vector Preparation

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The sFlt‐1 expression vector (pCMV‐sFlt‐1‐IRES‐bsr) was used as described previously.7 A VASH1 expression vector was prepared by inserting human VASH1 cDNA (NCBI database; Gene ID 22846) into the cloning site (Sma‐I, Xba‐I) of pCMV‐IRES‐bsr.17 SHIN‐3 and KOC‐2S were transfected with the sFlt‐1 or VASH1 expression vector, and a luciferase expression plasmid vector (pCMV‐LUC‐IRES‐bsr17) as a control, using Lipofectamine LTX and Plus Reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's instructions. The cells were then selectively cultured in a medium containing blasticidin S hydrochloride (Funakoshi Co., Ltd., Tokyo, Japan), and resistant cells were acquired.
Takahashi Y., Saga Y., Koyanagi T., Takei Y., Machida S., Taneichi A., Mizukami H., Sato Y., Matsubara S, & Fujiwara H. (2016). Vasohibin‐1 expression inhibits advancement of ovarian cancer producing various angiogenic factors. Cancer Science, 107(5), 629-637.
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5

Generating Stable Cell Lines for NK4, PTEN, and Luciferase

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The NK4, PTEN and luciferase (LUC) expression plasmid vectors that were used in the present study have been previously described (18 (link),32 (link)–35 (link)). These vectors were transfected into SKOV-3 using Lipofectamine-LTX and Plus reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The cells were selected using 10 μg/ml blasticidin S hydrochloride (Funakoshi Co., Ltd., Tokyo, Japan). Resistant clones were obtained after 4 weeks as SKOV-3/NK4, SKOV-3/PTEN and SKOV-3/LUC (control). The cells were subsequently maintained in the presence of 10 μg/ml blasticidin S hydrochloride.
WANG D., SAGA Y., SATO N., NAKAMURA T., TAKIKAWA O., MIZUKAMI H., MATSUBARA S, & FUJIWARA H. (2016). The hepatocyte growth factor antagonist NK4 inhibits indoleamine-2,3-dioxygenase expression via the c-Met-phosphatidylinositol 3-kinase-AKT signaling pathway. International Journal of Oncology, 48(6), 2303-2309.
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6

Cell Line Maintenance for Hybrid Artificial Chromosomes

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DT40 cells containing the tet-O HAC were cultured in Roswell Park Memorial Institute medium 1640 (RPMI-1640, GIBCO) supplemented with 10% fetal bovine serum (Biowest, Nuaille, France), 1% chicken serum (Invitrogen), 50 µM 2-melcaptometanol (SIGMA) and 15 µg/ml Blasticidin S hydrochloride (Funakoshi, Tokyo, Japan) at 40°C in 10% CO2. After introduction of the DsRed gene, CHO cells retaining the tet-O-DsRed-HAC were maintained in Ham's F-12 nutrient mixture (Invitrogen) plus 10% fetal bovine serum with 800 µg/ml G418 (Calbiochem). HT1080 cells containing the EGFP-21HAC were cultured in Dulbecco's Modified Eagle's Medium (DMEM, SIGMA) supplemented with 10% fetal bovine serum (Biowest, Nuaille, France) and with 4 µg/ml Blasticidin S at 37°C in 5% CO2. After transfer of the tet-O-DsRed-HAC, HT1080 cells containing both HACs were cultured with 4 µg/ml Blasticidin S and 400 µg/ml G418.
Iida Y., Kazuki Y., Hayashi M., Ueda Y., Hasegawa M., Kouprina N., Larionov V, & Oshimura M. (2014). A bi-HAC vector system towards gene and cell therapy. ACS synthetic biology, 3(2), 83-90.
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