For the culture of ES cells, the MEF were irradiated at 60 Gy and then plated on the gelatinized plates. Irradiated MEFs (26105 cells) were coated on the 6 well plates to support theSomatic Cell Nuclear Transfer (SCNT), Embryo Culture and Derivation of p182/2 ES Cell Lines
SCNT was performed by direct nuclear injection as previously reported [41]. In brief, BDF1 (C57BL/6 X DBA/2) mice from Charles River Laboratories (Wilmington, MA) were used as the oocyte donors, and p182/2 GFP (C57BL/6) mice were used as nuclei donors. Nuclei of p182/2 GFP BM cells were directly injected into enucleated eggs. The reconstructed oocytes were cultured in CZB medium for 1? h and further placed into calcium-free CZB medium containing 10 mM strontium and 5 mg/ml cytochalasin B for 6 h of activation treatment. Activated oocytes were cultured in KSOM+ AA medium for 4 days at 37uC in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2.
Figure 5. Reassortment of CDKI to CDKs by p18 overexpression in ES cells. (A, B and C) Real-time RT-PCR analysis of p21, p27, and CDK2 mRNA levels. All values were normalized to b-actin. Values are expressed as the mean 6 SD. (D) Western blotting performed to detect protein levels of p18, CDK2, CDK4, CDK6, pRb-Ser795, pRb-Ser811, and total Rb. Detection of b-actin was used as a loading control. (E) IP assays of p18, p21 and p27 were performed using cell lysate (100 mg total protein) from stably transduced p18, or WT ES cells. Immunocomplexes obtained were then immunoblotted with anti-CDK4 and anti-CDK2 antibodies. (F) Total protein extracts were obtained from stably transduced p18, or WT ES cells and immunoblotted with anti-p18, anti-p21, anti-p27, and anti-cdk2 and anti-cdk4 antibodies. b-actin was used as a loading control.
(G) A model for a proposed mechanism by which p18 enhances the self-renewal of ES cells, while inhibiting their differentiation potential. Briefly, ectopic expression of p18 promotes overexpression of CDK4, which in turn enhances the association of p21 and p27 with CDK4, and ultimately upregulates CDK2 activities. As a result, the cell cycle is accelerated and the self-renewal is enhanced, whereas the differentiation process is slowed. In A to F, data represent three independent experiments with similar results. When cloned p182/2 GFP embryos reached the blastocyst stage after 4 days of culture, they were transferred into plates containing inactive primary mouse embryonic fibroblast (pMEF) feeder cells in ESC medium and cultured for about 7?0 days [42]. The ESC medium used for ESC derivation including Knockout-DMEM (Invitrogen, Carlsbad, CA) supplemented with 15% knockout serum replacement (KSR, Invitrogen), 1,000 U/ml leukemia inhibitory factor (LIF) (Invitrogen), 1% penicillin-streptomycin (Invitrogen), 1% L-glutamine (Specialty Media), 1% non-essential amino acids (Specialty Media), 1% nucleosides for ES cells (Specialty Media), 1% 2-mercaptoethanol (Specialty Media) and 6 mM PD98059 (Promega, Madison, WI). Newly formed inner cell mass outgrowths were mechanically dissociated using trypsin (Invitrogen, Carlsbad, CA) treatment and replated on pMEF feeder cells until stable cell lines established. This work was performed in Dr Jerry Yang’s laboratory at the University of Connecticut.
Chimera Formation
The p182/2 ES cells were microinjected into ICR host blastocysts and transferred into 2.5-day post-coitum ICR pseudopregnant recipient females. Chimaerism was ascertained after birth by the appearance of black coat color (from p182/2 ES cells) in white host pups.
Lentiviral Vector Constructs
The iDuet101 lentiviral vector was kindly gifted by Dr. Linzhao Cheng (Johns Hopkins University). The vector constructs were made by inserting the full length p18 cDNA into iDuet101 by Kpn I digestion to get iDuet101-p18-GFP or by Cla I and Kpn I digestion to replace the GFP and get iDuet101-p18 (Fig 1G).
Lentiviral Vector Production
Lipofectamine (Invitrogen, Carlsbad, California) transfection was used to generate the virus supernatant. In brief, 293T cells were transfected using the lipofectamine transfection reagent with the cytomegalovirus (CMV) R8.91 and pMD.G helper plasmids. The lipofectamine-DNA mixture was applied directly onto 293T cells. The medium was replaced at 20?4 hours post-transfection and the medium containing the viral particles was collected at 48 hours post-transfection, filtered through 0.45-mm filters for use and supplemented with 6-mg/ml polybrene and Leukemia inhibitory factor (LIF).
Lentiviral Transduction on ES Cells
Mouse ES cells were grown on a feeder layer of irradiated MEFs in the presence of LIF. ES cells used for viral infection were washed, trypsinized and plated at a density of 106 cells in the wells of a 6-well gelatin-coated dish, and viral supernatant was added for 4 hr to overnight in the presence of 5 mg/ml polybrene (Sigma, St Louis, MO) and LIF. Following viral infection, the ES cells were resuspended in fresh ES cell medium and grown on a new feeder layer of irradiated MEFs. At three days post-infection, hygromycin B was added to select for p18 overexpression ES cells and GFP positive cells.
EDTA solution and washed with PBS three times. EBs were also harvested and washed 3 times with PBS. Total RNA was extracted by using RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. RNA was treated with RNaseree DNase (Invitrogen, Carlsbad, California) for 15 min at room temperature before reverse transcription with superscript II RT (Invitrogen, Carlsbad, California). Real- time PCR was performed on the chromo 4TM detector (M J Research, Waltham, MA) with SYBR Green PCR master mix (DyNAmo TM HS distributed by New England Biolabs Ipswich, MA). PCR conditions consisted of a 10-min hot start at 95uC followed by 40 cycles of 95uC for 15 sec, 60uC for 1 min and incubation for 3 sec at 77uC with a final extension for 10 min at 72uC. The average threshold cycle (Ct) for each gene was determined from triplicate reactions and the levels of gene expression relative to b-actin were determined as described [44]. Gene expression analysis for ES markers (Oct4, Sox2, Nanog, Rex1, Sall4) and differentiation markers (Cdx2, Brachyury, Gata6, Map2) were performed using published primers [43] (Table 1).
