Microsurgical analysis of the clonal age and the cell-cycle stage required for the onset of autogamy in Paramecium tetraurelia

Cell cycle stage of donor cells significantly influences the cloning efficiency during SCNT. Donor cells in G1/G0 stage have better capability to undergo nuclear reprogramming following transfer to an unfertilized oocyte. The lack of availability of cells synchronized at G1/G0 stage is one of the major factors limiting cloning efficiency in buffalo. The aim of this study was to compare the efficacy of various methods for cell cycle synchronization of buffalo fetal ﬁbroblast cells for SCNT. Cells isolated from fetus, 2 to 3 months old, were cultured in DMEM + 10% FBS. The primary culture was sub-cultured 8 to 10 times. For cell cycle synchronization, the cells were cultured to 1) 60 to 70% confluence (controls), 2) 60 to 70% confluence followed by serum starvation (DMEM + 0.5% FBS) for 24 h (serum starved), 3), full confluence followed by culture for additional 3 to 5 days (full confluent), 4) full confluence followed by serum starvation (DMEM + 0.5% FBS) for 24 h (full confluent+serum starved) and 5) 60 to 70% confluence followed by treatment with roscovitine (10, 20, or 30 μM) for 24 h. The synchronization efficiency was examined by propidium iodide staining followed by analysis of DNA content using flow cytometry and the data were analysed by 1-way ANOVA followed by Fisher’s l.s.d. test after arcsine transformation. The percentage of cells in G0/G1 phase of cell cycle was significantly higher (P < 0.05) in the full confluent+serum starved and roscovitine treated (20 or 30 μM) groups than that in the full confluent group and that treated with 10 μM roscovitine which, in turn, was higher (P < 0.05) than that in the serum starved and control groups. These results suggest that buffalo fetal fibroblast cells can be synchronized by roscovitine treatment or by serum starvation of fully confluent cell cultures to obtain a high proportion of cells in G0/G1 stage for SCNT. Table 1.Buffalo skin fibroblast cells at various stages following different treatments for cell cycle synchronization Supported by grant No. 1(5)/2007-NAIP from ICAR, India.

Download Full-text

Developmental competence of in vitro-fertilized porcine oocytes after in vitro maturation and solid surface vitrification: Effect of cryopreservation on oocyte antioxidative system and cell cycle stage

Cryobiology ◽

10.1016/j.cryobiol.2007.06.008 ◽

2007 ◽

Vol 55 (2) ◽

pp. 115-126 ◽

Cited By ~ 116

Author(s):

Tamás Somfai ◽

Manabu Ozawa ◽

Junko Noguchi ◽

Hiroyuki Kaneko ◽

Ni Wayan Kuriani Karja ◽

...

Keyword(s):

Cell Cycle ◽

Solid Surface ◽

In Vitro Maturation ◽

Developmental Competence ◽

Antioxidative System ◽

Cell Cycle Stage

Download Full-text

Histone proteins in HeLa S3 cells are synthesized in a cell cycle stage specific manner

Science ◽

10.1126/science.7058333 ◽

1982 ◽

Vol 215 (4533) ◽

pp. 683-685 ◽

Cited By ~ 36

Author(s):

F Marashi ◽

L Baumbach ◽

R Rickles ◽

F Sierra ◽

J. Stein ◽

...

Keyword(s):

Cell Cycle ◽

Histone Proteins ◽

Cell Cycle Stage ◽

Specific Manner ◽

A Cell ◽

Hela S3 ◽

Hela S3 Cells

Download Full-text

Cell cycle stage-specific transcription of histone genes

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(75)90660-9 ◽

1975 ◽

Vol 63 (4) ◽

pp. 945-949 ◽

Cited By ~ 24

Author(s):

G.S. Stein ◽

W.D. Park ◽

C.L. Thrall ◽

R.J. Mans ◽

J.L. Stein

Keyword(s):

Cell Cycle ◽

Histone Genes ◽

Cell Cycle Stage

Download Full-text

Transformation of isolated barley (Hordeum vulgare L.) microspores: I. The influence of pretreatments and osmotic treatment on the time of DNA synthesis

Genome ◽

10.1139/g08-112 ◽

2009 ◽

Vol 52 (2) ◽

pp. 166-174 ◽

Cited By ~ 11

Author(s):

Youn-Seb Shim ◽

K. Peter Pauls ◽

Ken J. Kasha

Keyword(s):

Cell Cycle ◽

Hordeum Vulgare ◽

Transgenic Plants ◽

Dna Synthesis ◽

Particle Bombardment ◽

Osmotic Adjustment ◽

Hordeum Vulgare L ◽

Cell Cycle Stage ◽

Dna Density ◽

Homozygous Transgenic Plants

The objective of this study was to determine when DNA synthesis occurred during pretreatments of cultured barley ( Hordeum vulgare L.) microspores and during their preparation for particle bombardment. Based on this information, an investigation of the influence of cell cycle stage on the ability to obtain homozygous transgenic plants by particle bombardment will be presented in paper II of this series. It was hypothesized that the introduction of foreign genes at the G1 cell cycle stage in cultured uninucleate microspores would produce homozygous transgenic plants. Experiments were conducted with two different commonly used pretreatments to induce microspore embryogenesis: cold (4 °C) for 21days and cold plus 0.3 mol/L mannitol for 4 days. After pretreatment, the microspores were placed in a higher osmotic medium for 4 h prior to and for 18 h following bombardment. It was confirmed that during the cold plus mannitol pretreatment, there was no apparent change in the cell cycle stage, with the majority of the microspores remaining at the G1 stage. While in the cold for 21 days, the microspores progressed slowly through to G2, with a few progressing further into the mitosis and binucleate stages. Hourly DNA density measurements that were taken during the 4 h osmotic adjustment period following the cold plus mannitol pretreatment indicated that DNA synthesis began during this period at 25 °C, while at 4 °C, there was no apparent change in cell cycle stage or in DNA density. Thus, one might expect to find a higher frequency of homozygous doubled haploids by maintaining the temperature low during the 4 h osmotic adjustment period following the cold plus mannitol pretreatment than following the 21 day cold pretreatment. However, it is also not known what effect the temperatures during the whole high-osmotic treatments will have on the rate and time of incorporation of the transgene.

Download Full-text