Cell Colony Formation Induced by Xenopus Egg Extract as a Marker for Improvement of Cloned Blastocyst Formation in the Pig

2011 ◽  
Vol 13 (6) ◽  
pp. 521-526 ◽  
Author(s):  
Ying Liu ◽  
Olga Østrup ◽  
Juan Li ◽  
Gábor Vajta ◽  
Peter M. Kragh ◽  
...  
Keyword(s):  
Colony Formation ◽  
Blastocyst Formation ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg ◽  
Cell Colony

scholarly journals 21 EFFECT OF SECOND TIME XENOPUS EGG EXTRACT TREATMENT ON COLONY FORMATION AND CLONED BLASTOCYST FORMATION IN PIG

2012 ◽  
Vol 24 (1) ◽  
pp. 122 ◽  
Author(s):  
Y. Liu ◽  
O. Østrup ◽  
R. Li ◽  
G. Vajta ◽  
P. M. Kragh ◽  
...  
Keyword(s):  
Colony Formation ◽  
Cultured Cells ◽  
Activity Level ◽  
Blastocyst Formation ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Fetal Fibroblasts ◽  
Colony Number ◽  
Xenopus Egg ◽  
Blastocyst Rate

Extract from Xenopus eggs can induce reprogramming in somatic cells. In our previous study, cell colony formation was induced during culture of porcine fetal fibroblasts after a single treatment with Xenopus egg extract and culture for several passages and using these long-term cultured cells for cloning increased the resulting blastocyst rate (Liu et al. 2011 Reprod. Fertil. Dev. 23, 130). However, both colony number and cloned blastocyst rate decreased after Passage 15 and no colonies formed after Passage 18. Therefore, in this study we investigated the effect of a second extract treatment on colony formation and cloned blastocyst formation. Extract-treated (ExT) porcine fetal fibroblasts at Passage 13 (P13) grown on poly-L-lysine-coated coverslips were permeabilized by digitonin (7 μg mL–1, 2 min, 4°C) and incubated in extract at 37°C for 30 min. After resealing the membrane in DMEM supplemented with 2 mM CaCl2, the remaining cells were cultured in ES medium (Vejlsted et al. 2005 Mol. Reprod. Dev. 70, 445). The treated cells were split onto 2 coverslips on Day 7 after the second extract treatment (2ExT), defined as Passage 1 (2ExT P1, comparable with ExT P14). New subcultures were made every 7 to 8 days when 70 to 80% clusters became colonies (i.e. 2ExT P8). Colony cells from both ExT (P14 and P16) and 2ExT (P1, P3 and P6) were used for handmade cloning and nontreated cells were used as control (Day 0). Blastocyst rates were analysed by chi-square test and colony numbers were analysed by 1-way ANOVA (SAS version 9.2). Colony numbers and cloned blastocyst rates on Day 6 are summarised in Table 1. Colonies continued to form in treated cells from 2ExT P1 to P8. The colony number maintained at a high level (60 to 80) from 2ExT P4 to P8 and it was significantly higher than that of ExT cells at the comparable passage numbers. No colonies formed in control cells. When using 2ExT colony cells at P3 and P6 for cloning, the blastocyst rates increased compared with controls and they were also higher than in the ExT group. Cloned blastocyst rates were not different between 2ExT P1 and ExT P14 groups. In conclusion, a second extract treatment can induce colony formation and increase cloned blastocyst rates, indicating that this repeated extract treatment again could activate the extract-treated cells to an activity level similar to that achieved after the first treatment. Table 1.Summary of colony number and cloned blastocyst rate with ExT and 2ExT colony cells

Aphidicolin-Sensitive DNA Polymerase Is Incorporated into the Chromatin during Nuclear Envelope Assembly in Xenopus Egg Extract

1995 ◽  
Vol 219 (1) ◽  
pp. 283-291
Author(s):  
Yoshihiro Takasuga ◽  
Machiko Murata ◽  
Jinpei Yamashita ◽  
Toshiow Andoh ◽  
Tatsuo Yagura
Keyword(s):  
Nuclear Envelope ◽  
Dna Polymerase ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Nuclear Envelope Assembly ◽  
Xenopus Egg

scholarly journals Xenopus egg extract: A powerful tool to study genome maintenance mechanisms

2017 ◽  
Vol 428 (2) ◽  
pp. 300-309 ◽  
Author(s):  
Wouter S. Hoogenboom ◽  
Daisy Klein Douwel ◽  
Puck Knipscheer
Keyword(s):  
Genome Maintenance ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg

273 SELECTION OF PLURIPOTENT STEM CELLS INDUCED BY XENOPUS EGG EXTRACTS

2009 ◽  
Vol 21 (1) ◽  
pp. 234 ◽  
Author(s):  
C.-Y. Chiang ◽  
P.-C. Tang
Keyword(s):  
Somatic Cells ◽  
Egfp Expression ◽  
3T3 Cells ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

It has been reported that Xenopus egg extracts contain molecules that are capable of reprogramming mammalian somatic cells. The reprogrammed somatic cells, which are called extract treated cells (ETC), possess the potential for clinical therapy as embryonic stem (ES) cells do. Therefore, in addition to establishment of an efficient method to reprogram mouse NIH/3T3 cells by Xenopus egg extracts, the aim of this study was to select the ETC cells by the expression of Oct4. In Experiment 1, two methods, electroporation or permeabilization, were conducted to treat mouse NIH/3T3 cells with Xenopus egg extracts. 2 × 105 cells in 200 μL reprogramming mixture containing Xenopus egg extracts were stimulated by a direct current (DC) pulse (80 V mm–1 for 3 msec) three times followed by a pause of incubation at 37°C for 5 min and a single DC pulse (170 V mm–1, for 0.4 msec) subsequently. The electroporated cells were then incubated at 22°C for 1 h. In the other treatment group, NIH/3T3 cells (5 × 105) were permeabilized by streptolysin O (SLO, 500 ng mL–1 in PBS) for 50 min at 37°C before mixed with Xenopus egg extracts at 22°C for 2 h. Cells were cultured in DMEM supplemented with 10% FBS for the first 4 days and then changed to ES medium (DMEM supplemented with 15% FBS, 0.1 mm β-mercaptoethanol, 1000 unit mL–1 mLIF, 0.5% nonessential amino acids, 2 mm L-glutamine) for the last 6 days after Xenopus egg extract treatment. Cell colonies were found in both treatment groups at the end of culture. Examination by immunocytochemical staining, results showed that the extract-treated cell colonies expressed pluripotent marker proteins, such as alkaline phosphatase, Oct4, Nanog and Sox2. In Experiment 2, an enhanced green fluorescent protein (EGFP) expression vector was constructed and EGFP was driven by Oct4 enhancer and promoter (Oct4-EGFP). Mouse NIH/3T3 cells were then transfected with Oct4-EGFP plasmids and selected for stable clone by G418 screening. After 6 passages, the NIH/3T3-Oct4-EGFP cells were treated with egg extracts to induce reprogramming as Experiment 1, and monitored pluripotency based on the expression of EGFP. Results showed that some of the cells or cell colonies expressed green fluorescence driven by Oct4 regulatory element at the 8th day of culture after extract treatment. Our results demonstrated that both methods of electroporation and reversible permeabilization could introduce reprogramming molecules in Xenopus egg extract to the mammalian somatic cells and generate ETCs cells in vitro. Also, with the establishment of NIH/3T3-Oct4-EGFP cell line, the potentially reprogrammed colonies could be easily selected by EGFP expression. The changes of epigenetic modifications in the ETC cells would be investigated in the short future.

scholarly journals Replication Origins in XenopusEgg Extract Are 5–15 Kilobases Apart and Are Activated in Clusters That Fire at Different Times

2001 ◽  
Vol 152 (1) ◽  
pp. 15-26 ◽  
Author(s):  
J. Julian Blow ◽  
Peter J. Gillespie ◽  
Dennis Francis ◽  
Dean A. Jackson
Keyword(s):  
Dna Sequence ◽  
Replication Origins ◽  
Origin Firing ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Sperm Nuclei ◽  
Dna Replication Origins ◽  
Small Clusters

When Xenopus eggs and egg extracts replicate DNA, replication origins are positioned randomly with respect to DNA sequence. However, a completely random distribution of origins would generate some unacceptably large interorigin distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [3H]thymidine or bromodeoxyuridine and the geometry of labeled sites on spread DNA was examined. Most origins were spaced 5–15 kb apart. This regular distribution provides an explanation for how complete chromosome replication can be ensured although origins are positioned randomly with respect to DNA sequence. Origins were grouped into small clusters (typically containing 5–10 replicons) that fired at approximately the same time, with different clusters being activated at different times in S phase. This suggests that a temporal program of origin firing similar to that seen in somatic cells also exists in the Xenopus embryo. When the quantity of origin recognition complexes (ORCs) on the chromatin was restricted, the average interorigin distance increased, and the number of origins in each cluster decreased. This suggests that the binding of ORCs to chromatin determines the regular spacing of origins in this system.

scholarly journals Ubiquitin-RAS peptide extensions as substrates for farnesyl-protein transferase and carboxymethyltransferase

1992 ◽  
Vol 285 (1) ◽  
pp. 55-60 ◽  
Author(s):  
Y Yoo ◽  
S Watts ◽  
M Rechsteiner
Keyword(s):  
Heat Treatment ◽  
Binding Assay ◽  
Rabbit Reticulocyte Lysate ◽  
Farnesyl Pyrophosphate ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Farnesyl Protein Transferase ◽  
Reticulocyte Lysate ◽  
One Step ◽  
Xenopus Egg

Using oligonucleotide-mediated ‘loop-in’ mutagenesis strategies in M13, a heat-inducible ubiquitin (Ub) gene was extended by sequences coding for the C-terminal 11 amino acids of Ha-RAS. The resulting gene was transformed into AR13 and production of the Ub-peptide extension was induced by heat treatment. After one-step purification, the fusion protein (Ub-cRAS) was used as a substrate for farnesyl-protein transferase. Ub-cRAS was farnesylated on incubation in Xenopus egg extract or rabbit reticulocyte lysate. In contrast, when serine was substituted for the last cysteine in the RAS extension, transfer of the [3H]farnesyl group from [3H] farnesyl pyrophosphate to the modified Ub-cRAS was not observed. Farnesylation of Ub-cRAS permitted us to develop an easy membrane-binding assay for farnesyl-protein transferase enzyme activity. Using this assay, we partially purified the enzyme from rabbit reticulocyte lysate. We also detected methylation of the farnesylated Ub-cRAS terminus in Xenopus egg extract.

scholarly journals Spatial Variation of Microtubule Depolymerization in Large Asters

2021 ◽  
pp. mbc.E20-11-0723
Author(s):  
Keisuke Ishihara ◽  
Franziska Decker ◽  
Paulo Caldas ◽  
James F. Pelletier ◽  
Martin Loose ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Microtubule Assembly ◽  
Microtubule Depolymerization ◽  
Physiological Regulation ◽  
Xenopus Egg Extract ◽  
Spatial Regulation ◽  
Important Target ◽  
Egg Extract ◽  
Xenopus Egg ◽  
Difference Imaging

Microtubule plus end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in  Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were higher in the aster interior compared to the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and MAPs in the interior cytosol compared to that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

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