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Xenopus egg extract is a lysate that is prepared by crushing the eggs of the African clawed frog Xenopus laevis. It offers a powerful cell-free (or in vitro) system for studying various cell biological processes, including cell cycle progression, nuclear transport, DNA replication and chromosome segregation. It is also called Xenopus egg cell-free system or Xenopus egg cell-free extract.

History

The first frog egg extract was reported in 1983 by Lohka and Masui. [1] This pioneering work used eggs of the Northern leopard frog Rana pipiens to prepare an extract. Later, the same procedure was applied to eggs of Xenopus laevis, becoming popular for studying cell cycle progression and cell cycle-dependent cellular events. [2] Extracts derived from eggs of the Japanese common toad Bufo japonicus [3] or of the Western clawed frog Xenopus tropicalis [4] have also been reported.

Basics of extract preparation

The cell cycle of unfertilized eggs of X. laevis is arrested highly synchronously at metaphase of meiosis II. Upon fertilization, the metaphase arrest is released by the action of Ca2+ ions released from the endoplasmic reticulum, thereby initiating early embryonic cell cycles that alternates S phase ( DNA replication) and M phase ( mitosis). [5]

M phase extract

Figure 1. An egg extract is prepared by crushing X. laevis eggs by centrifugation

Unfertilized eggs in a buffer containing the Ca2+ chelator EGTA (ethylene glycol tetraacetic acid) are packed into a centrifuge tube. After removing excess buffer, the eggs are crushed by centrifugation (~10,000 g). A soluble fraction that appears between the lipid cap and the yolk is called an M phase extract. This extract contains a high level of cyclin B- Cdk1. When demembranated sperm nuclei are incubated with this extract, it undergoes a series of structural changes and is eventually converted into a set of M phase chromosomes with bipolar spindles.

Interphase (S phase) extract

Different types of egg extracts

Cycling extract

Figure 2. An interphase nucleus (left) and a cluster of mitotic chromosomes (right) produced in a cycling extract. Bar, 10 μm.

High-speed supernatant (HSS)

Nucleoplasmic extract (NPE)

Discoveries made using egg extracts

More recently, the egg extracts have been used to study reprogramming of differentiated nuclei, [17] physical properties of spindles [18] and nuclei, [19] and theoretical understanding of cell cycle control. [20]

See also

References

  1. ^ Lohka MJ, Masui Y (1983). "Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components". Science. 220 (4598): 719–721. Bibcode: 1983Sci...220..719L. doi: 10.1126/science.6601299. PMID  6601299.
  2. ^ Lohka MJ, Maller JL. (1985). "Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts". J. Cell Biol. 101 (2): 518–523. doi: 10.1083/jcb.101.2.518. PMC  2113692. PMID  3926780.
  3. ^ Ohsumi K, Katagiri C (1991). "Characterization of the ooplasmic factor inducing decondensation of and protamine removal from toad sperm nuclei: involvement of nucleoplasmin". Dev. Biol. 148 (1): 295–305. doi: 10.1016/0012-1606(91)90338-4. PMID  1936566.
  4. ^ Brown KS, Blower MD, Maresca TJ, Grammer TC, Harland RM, Heald R (2007). "Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle". J. Cell Biol. 176 (6): 765–770. doi: 10.1083/jcb.200610043. PMC  2064050. PMID  17339377.{{ cite journal}}: CS1 maint: multiple names: authors list ( link)
  5. ^ Masui Y (2000). "The elusive cytostatic factor in the animal egg". Nat. Rev. Mol. Cell Biol. 1 (3): 228–232. doi: 10.1038/35043096. PMID  11252899. S2CID  5303121.
  6. ^ Lohka MJ, Hayes MK, Maller JL (1988). "Purification of maturation-promoting factor, an intracellular regulator of early mitotic events". Proc. Natl. Acad. Sci. USA. 85 (9): 3009–3013. Bibcode: 1988PNAS...85.3009L. doi: 10.1073/pnas.85.9.3009. PMC  280132. PMID  3283736.{{ cite journal}}: CS1 maint: multiple names: authors list ( link)
  7. ^ Murray AW, Kirschner MW (1989). "Cyclin synthesis drives the early embryonic cell cycle". Nature. 339 (6222): 275–280. Bibcode: 1989Natur.339..275M. doi: 10.1038/339275a0. PMID  2566917. S2CID  4352582.
  8. ^ Murray AW, Solomon MJ, Kirschner MW (1989). "The role of cyclin synthesis and degradation in the control of maturation promoting factor activity". Nature. 339 (6222): 280–286. Bibcode: 1989Natur.339..280M. doi: 10.1038/339280a0. PMID  2566918. S2CID  4319201.{{ cite journal}}: CS1 maint: multiple names: authors list ( link)
  9. ^ Holloway SL, Glotzer M, King RW, Murray AW (1993). "Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor". Cell. 73 (7): 1393–1402. doi: 10.1016/0092-8674(93)90364-v. PMID  8391932. S2CID  26338475.{{ cite journal}}: CS1 maint: multiple names: authors list ( link)
  10. ^ Heald R, Tournebize R, Blank T, Sandaltzopoulos R, Becker P, Hyman A, Karsenti E (1996). "Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts". Nature. 382 (6590): 420–425. Bibcode: 1996Natur.382..420H. doi: 10.1038/382420a0. PMID  8684481. S2CID  4238425.{{ cite journal}}: CS1 maint: multiple names: authors list ( link)
  11. ^ Blow JJ, Laskey RA (1988). "A role for the nuclear envelope in controlling DNA replication within the cell cycle". Nature. 332 (6164): 546–548. Bibcode: 1988Natur.332..546B. doi: 10.1038/332546a0. PMID  3357511. S2CID  4313693.
  12. ^ Kubota Y, Mimura S, Nishimoto S, Takisawa H, Nojima H (1995). "Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor". Cell. 81 (4): 601–609. doi: 10.1016/0092-8674(95)90081-0. PMID  7758114. S2CID  18797719.{{ cite journal}}: CS1 maint: multiple names: authors list ( link)
  13. ^ Görlich D, Prehn S, Laskey RA, Hartmann E (1994). "Isolation of a protein that is essential for the first step of nuclear protein import". Cell. 79 (5): 767–778. doi: 10.1016/0092-8674(94)90067-1. PMID  8001116. S2CID  7539929.{{ cite journal}}: CS1 maint: multiple names: authors list ( link)
  14. ^ Hirano T, Mitchison TJ (1994). "A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro". Cell. 79 (3): 449–458. doi: 10.1016/0092-8674(94)90254-2. PMID  7954811. S2CID  24140495.
  15. ^ Hirano T, Kobayashi R, Hirano M (1997). "Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein". Cell. 89 (4): 511–521. doi: 10.1016/s0092-8674(00)80233-0. PMID  9160743. S2CID  15061740.{{ cite journal}}: CS1 maint: multiple names: authors list ( link)
  16. ^ Losada A, Hirano M, Hirano T (1998). "Identification of Xenopus SMC protein complexes required for sister chromatid cohesion". Genes Dev. 12 (13): 1986–1997. doi: 10.1101/gad.12.13.1986. PMC  316973. PMID  9649503.{{ cite journal}}: CS1 maint: multiple names: authors list ( link)
  17. ^ Ganier O, Bocquet S, Peiffer I, Brochard V, Arnaud P, Puy A, Jouneau A, Feil R, Renard JP, Méchali M (2011). "Synergic reprogramming of mammalian cells by combined exposure to mitotic Xenopus egg extracts and transcription factors". Proc Natl Acad Sci USA. 108 (42): 17331–17336. doi: 10.1073/pnas.1100733108. PMC  3198361. PMID  21908712.{{ cite journal}}: CS1 maint: multiple names: authors list ( link)
  18. ^ Shimamoto Y, Maeda YT, Ishiwata S, Libchaber AJ, Kapoor TM (2011). "Insights into the micromechanical properties of the metaphase spindle". Cell. 145 (7): 767–778. doi: 10.1016/j.cell.2011.05.038. PMC  3124677. PMID  21703450.{{ cite journal}}: CS1 maint: multiple names: authors list ( link)
  19. ^ Hara Y, Merten CA (2015). "Dynein-based accumulation of membranes regulates nuclear expansion in Xenopus laevis egg extracts". Dev Cell. 33 (5): 562–575. doi: 10.1016/j.devcel.2015.04.016. PMID  26004509.
  20. ^ Pomerening JR, Kim SY, Ferrell JE Jr (2005). "Systems-level dissection of the cell-cycle oscillator: bypassing positive feedback produces damped oscillations". Cell. 122 (4): 565–578. doi: 10.1016/j.cell.2005.06.016. PMID  16122424. S2CID  11835940.{{ cite journal}}: CS1 maint: multiple names: authors list ( link)