Pd03259010

Manufactured by Selleck Chemicals

The PD03259010 is a laboratory equipment product designed for general scientific and analytical applications. It serves as a versatile tool for researchers and laboratory professionals. The core function of this product is to provide a reliable and accurate method for data collection and analysis. Further details on the intended use or specific applications of this product are not available.

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8 protocols using pd03259010

Generation of Gene-Edited Haploid Mouse Embryonic Stem Cells

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Mouse DKO-AG-haESCs (IG^ΔDMR-H19^ΔDMR-AGH-2 cells or O48 cells) [24 (link)] were cultured in ESC medium containing 15% FBS (Excell Bio), penicillin–streptomycin, non-essential amino acids, Nucleosides, l-glutamine, β-mercaptoethanol, 1000 U/ml Lif, 1 μM PD03259010 (Selleck), and 3 μM CHIR99021 (Selleck) in DMEM (Gibco) at 37 ℃ and 5% CO₂. As previously described [23 (link), 24 (link)], DKO-AG-haESCs were passaged every 2–3 days by trypsinization, and haploid cells were enriched every 2–3 weeks by fluorescence-activated cell sorting (FACS). One million AG-haESCs were passaged into a well of a six-well plate and then transfected with 2 μg CRISPR-Cas9 plasmids (pX330-mCherry-35delG or pX330-mCherry-235delC) and 1 μg pMD19T vectors (19 T-Gjb2-35delG or 19 T-Gjb2-235delC, respectively) using Lipofectamine 3000 (Life Technologies) following the manufacturer’s instructions. After 18–24 h, mCherry-positive haploid cells were enriched by FACS and then 6000–10,000 cells were seeded into a new well of a six-well plate. After 6–8 days, single clones derived from a single cell were picked and genotyped using PCR and Sanger sequencing.

Li Q., Cui C., Liao R., Yin X., Wang D., Cheng Y., Huang B., Wang L., Yan M., Zhou J., Zhao J., Tang W., Wang Y., Wang X., Lv J., Li J., Li H, & Shu Y. (2023). The pathogenesis of common Gjb2 mutations associated with human hereditary deafness in mice. Cellular and Molecular Life Sciences, 80(6), 148.

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Haploid Embryonic Stem Cell Culture and Injection

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A detailed protocol of AG-haESCs culture and intracytoplasmic AG-haESC injection (ICAHCI) has been described in previous works^{59 (link),60 (link)}. Briefly, mouse AG-haESCs (including WT- and DKO-AG-haESCs) were cultured in DMEM with 15% FBS (Excell Bio), penicillin–streptomycin, non-essential amino acids, NUC, l-glutamine, β-mercaptoethanol, 1000 U ml⁻¹ Lif, 1μM PD03259010 (Selleck) and 3μM CHIR99021 (Selleck). sgRNAs for different miRNA deletions were synthesized and ligated in pX330-mCherry plasmid^{69 (link)}. Constructed plasmids were then transfected into O48, which were enriched haploid cells (Hoechst 33342 staining) and mCherry positive cells through FACS after 24 h. Gene-modified cell lines were obtained from single-cell expansion and genotyping, which were then subjected to the ICAHCI experiment.

Li Q., Lu J., Yin X., Chang Y., Wang C., Yan M., Feng L., Cheng Y., Gao Y., Xu B., Zhang Y., Wang Y., Cui G., Xu L., Sun Y., Zeng R., Li Y., Jing N., Xu G.L., Wu L., Tang F, & Li J. (2023). Base editing-mediated one-step inactivation of the Dnmt gene family reveals critical roles of DNA methylation during mouse gastrulation. Nature Communications, 14, 2922.

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Generation of HA-Tagged Haploid Human ESCs

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DKO-AG-haESCs were maintained in DMEM (Millipore) with 15% FBS (Gibco), penicillin/streptomycin, non-essential amino acids, l-glutamine, nucleosides, 2-mercaptoethanol, 1000 U/ml Lif, 1 μM PD03259010 (Selleck) and 3 μM CHIR99021 (Selleck). Cells were passaged every 2–3 days (23 (link)). DKO-AG-haESCs were transfected with CRISPR-Cas9 plasmid and HA tag KI DNA donor using Lipofectamine 2000 (Life Technologies). At 24 h after transfection, the mCherry-positive haploid cells were enriched by fluorescence-activated cell sorting (FACS, BD AriaII) and plated at a low density. In 7–8 days after plating, single colonies were picked up, and positive colonies for the HA tag were selected by genomic DNA PCR amplification for Sanger sequencing.

Liu H., Huang T., Li M., Li M., Zhang C., Jiang J., Yu X., Yin Y., Zhang F., Lu G., Luo M.C., Zhang L.R., Li J., Liu K, & Chen Z.J. (2019). SCRE serves as a unique synaptonemal complex fastener and is essential for progression of meiosis prophase I in mice. Nucleic Acids Research, 47(11), 5670-5683.

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Serum-Free Culture of Mouse ESCs

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mESCs were cultured in “Serum Free ES” (SFES) media supplemented with 2i. SFES media consists of 500 mL DMEM/F12 (Gibco #11320–033), 500 mL Neurobasal (Gibco #21103–049), 5 mL N2 Supplement (Gibco #17502–048), 10 mL B27 with retinoic acid (gibco #17504–044), 6.66 mL 7.5% BSA (Gibco #15260–037), 10 mL 100x GlutaMax (Gibco #35050–061), and 10 mL 100x Pen/Strep. To make “2i SFES”, 1 nM PD03259010 (Selleckchem #S1036), 3 nM CHIR99021 (Selleckchem #S2924) and 1000 units/mL LIF (ESGRO #ESG1106) were added to 45 mL SFES. Prior to use, 1-thioglycerol (MTG; Sigma M6145) was diluted 1.26% in SFES and added 1:1000 to 2i SFES media. To passage, mESCs were treated with 1 mL of accutase in a 6 well plate (Corning #353046) for 5 minutes at room temperature (RT). After incubation, cells were mixed by pipette and moved to a 15 mL conical tube, supplemented with 10 mL SFES and spun at 300xg for 3 minutes. Then, media was removed and cells were counted using the Countess II Cell Counter (ThermoFisher) according to the manufacturer’s instructions. Cells were then plated in 6 well plates that had gelatinized with 1% gelatin for 30 minutes at 37 C at 5 × 10^5 cells per well in 2 mL of 2i SFES. Media was changed every day and cells were split every other day. Cells and culturing reagents were gifted by Dr. Abigail Buchwalter.

Kim M.S., Bhargava H.K., Shavey G.E., Lim W.A., El-Samad H, & Ng A.H. (2024). Degron-based bioPROTACs for controlling signaling in CAR T cells. bioRxiv.

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Feeder-free Maintenance of Murine ESCs

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E14 mESCs (gift from the Panning lab, UCSF) were maintained on gelatin coated dishes in 2i+LIF media, composed of a 1:1 mixture of DMEM/F12 (Thermo Fisher, 11320–033) and Neurobasal (Thermo Fisher, 21103–049) supplemented with N2 supplement (Thermo Fisher, 17502–048), B27 with retinoid acid (Thermo Fisher, 17504–044), 0.05% BSA (Thermo Fisher, 15260–037), 2 mM GlutaMax (Thermo Fisher, 35050–061), 150 µM 1-thioglycerol (Sigma, M6145), 1 µM PD03259010 (Selleckchem, 1036), 3 µM CHIR99021 (Selleckchem, S2924) and 10⁶U/L leukemia inhibitory factor (Peprotech, 250–02).

Meyer K., Lammers N.C., Bugaj L.J., Garcia H.G, & Weiner O.D. (2023). Optogenetic control of YAP reveals a dynamic communication code for stem cell fate and proliferation. Nature Communications, 14, 6929.

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Maintenance of H19ΔDMR-IGΔDMR-AGH haESCs

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haESCs (H19ΔDMR-IGΔDMR-AGH) were maintained in a standard ESC culture system: DMEM (Millipore) with 15% FBS (Gibco), penicillin-streptomycin (Gibco), nucleosides (Millipore), non-essential amino acids (Millipore), L-glutamine (Millipore), β-mercaptoethanol (Millipore), 1,000 U/mL LIF(Millipore), 3 μM CHIR99021 (Selleck) and 1 μM PD03259010 (Selleck) ^{26 (link),51 (link)–53 (link)}.

Wang Y., Qu Z., Fang Y., Chen Y., Peng J., Song J., Li J., Shi J., Zhou J.Q, & Zhao Y. (2023). Chromosome territory reorganization through artificial chromosome fusion is compatible with cell fate determination and mouse development. Cell Discovery, 9, 11.

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Maintenance of 129/CastEiJ Hybrid Mouse ESCs

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129/CastEiJ F1 hybrid mouse embryonic stem cells were maintained in 2i + Lif media, composed of a 1:1 mixture of DMEM/F12 (Thermo Fisher Waltham, MA, #11320–033) and Neurobasal (Thermo Fisher #21103–049) supplemented with N2 supplement (Thermo Fisher #17502–048), B27 with retinoid acid (Thermo Fisher #17504–044), 0.05% BSA (Thermo Fisher #15260–037), 2 mM GlutaMax (Thermo Fisher #35050–061), 150 µM 1-thioglycerol (Sigma St. Louis, MO, M6145), 1 µM PD03259010 (Selleckchem Houston, TX, #1036), 3 µM CHIR99021 (Selleckchem #S2924) and 10⁶ U/L leukemia inhibitory factor (Peprotech Rocky Hill, NJ, #250–02). Media was changed daily and cells were passaged every 2 days.129/CastEiJ ESCs were genetically verified by PCR amplification and Sanger sequencing of regions within the Sox2 locus to identify predicted SNPs between the parental genomes. These cells tested negatively for mycoplasma using MycoAlert Detect Kit (Lonza Basal, Switzerland #LT07-318).

Alexander J.M., Guan J., Li B., Maliskova L., Song M., Shen Y., Huang B., Lomvardas S, & Weiner O.D. (2019). Live-cell imaging reveals enhancer-dependent Sox2 transcription in the absence of enhancer proximity. eLife, 8, e41769.

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Mouse Androgenetic Haploid ESCs Culture

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Mouse androgenetic haploid embryonic stem cells (AG-haESCs) of AGH-OG3 and H19 △DMR -IG △DMR -AGH-OG3 were from our previous works (Yang et al., 2012; Zhong et al., 2015) , respectively. These cells were cultured in DMEM (Merk) with 15% FBS (Thermo Scientific™), Penicillin-Streptomycin, Non Essential Amino Acids, Nucleosides, L-Glutamine Solution, 2-Mercaptoethanol, 1000 U/ml Lif, 1 μM PD03259010 (Selleck) and 3 μM CHIR99021 (Selleck). Cells were transfected using Lipofectamine 3000 reagent (Thermo Scientific™) according to the manufacturer's protocols.

Li Q., Li Y., Yin Q., Huang S., Wang K., Zhuo L., Li W., Chang B, & Li J. (2020). Temporal regulation of prenatal embryonic development by paternal imprinted loci. Science China. Life sciences, 63(1).

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