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Study of apoptosis in vitro using the Xenopus egg extract reconstitution system.

Publication ,  Journal Article
Deming, P; Kornbluth, S
Published in: Methods in molecular biology (Clifton, N.J.)
January 2006

It was first shown by Newmeyer and colleagues in the 1990s that the molecular events of apoptosis could be reconstituted in vitro using Xenopus egg extracts. When the egg extract is allowed to incubate at room temperature for an extended time, the biochemical events of apoptosis are activated spontaneously. The features of apoptosis in the Xenopus reconstitution system mimic those that occur in mammalian cells: Cytochrome c is released from the mitochondria, caspases are activated, cellular substrates are cleaved, and added nuclei are fragmented. Moreover, these apoptotic events can be inhibited by addition of antiapoptotic proteins such as Bcl-2 and conventional caspase inhibitors (e.g., ZVAD-fmk). The mitochondria, which are triggered to release cytochrome c by as-yet-unknown signals in the extract, are essential for induction of the spontaneous apoptotic program in these extracts. However, one can study the apoptotic events that occur downstream of mitochondrial cytochrome c release by preparing extracts devoid of membrane components (including mitochondria). When purified cytochrome c is added to such cytosolic extracts, biochemical markers of apoptosis (e.g., activation of caspases) can be monitored. The egg extract therefore offers a tractable system for studying either separately or together the events of apoptosis occurring upstream and downstream of the mitochondria.

Duke Scholars

Published In

Methods in molecular biology (Clifton, N.J.)

DOI

EISSN

1940-6029

ISSN

1064-3745

Publication Date

January 2006

Volume

322

Start / End Page

379 / 393

Related Subject Headings

  • Xenopus
  • Ovum
  • Male
  • Female
  • Developmental Biology
  • Cytochromes c
  • Cell Nucleus
  • Cell Extracts
  • Apoptosis
  • Animals
 

Citation

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Deming, P., & Kornbluth, S. (2006). Study of apoptosis in vitro using the Xenopus egg extract reconstitution system. Methods in Molecular Biology (Clifton, N.J.), 322, 379–393. https://doi.org/10.1007/978-1-59745-000-3_27
Deming, Paula, and Sally Kornbluth. “Study of apoptosis in vitro using the Xenopus egg extract reconstitution system.Methods in Molecular Biology (Clifton, N.J.) 322 (January 2006): 379–93. https://doi.org/10.1007/978-1-59745-000-3_27.
Deming P, Kornbluth S. Study of apoptosis in vitro using the Xenopus egg extract reconstitution system. Methods in molecular biology (Clifton, NJ). 2006 Jan;322:379–93.
Deming, Paula, and Sally Kornbluth. “Study of apoptosis in vitro using the Xenopus egg extract reconstitution system.Methods in Molecular Biology (Clifton, N.J.), vol. 322, Jan. 2006, pp. 379–93. Epmc, doi:10.1007/978-1-59745-000-3_27.
Deming P, Kornbluth S. Study of apoptosis in vitro using the Xenopus egg extract reconstitution system. Methods in molecular biology (Clifton, NJ). 2006 Jan;322:379–393.

Published In

Methods in molecular biology (Clifton, N.J.)

DOI

EISSN

1940-6029

ISSN

1064-3745

Publication Date

January 2006

Volume

322

Start / End Page

379 / 393

Related Subject Headings

  • Xenopus
  • Ovum
  • Male
  • Female
  • Developmental Biology
  • Cytochromes c
  • Cell Nucleus
  • Cell Extracts
  • Apoptosis
  • Animals