Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal – Via microinjection of a nucleus into an embryo – egg cell – or...
Reexamination Certificate
1998-06-18
2001-04-03
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
Via microinjection of a nucleus into an embryo, egg cell, or...
C435S325000, C435S375000
Reexamination Certificate
active
06211429
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the fields of animal husbandry and biomedicine. More particularly, the present invention relates to methods for improving the cloning of mammals.
2. Description of Related Art
Induction of Calcium Transients in Oocytes
Sperm-oocyte interaction triggers an increase in the intracellular free Ca
2+
concentration ([Ca
2+
]
i
) in oocytes, which in mammals takes the form of a series of repetitive Ca
2+
transients [1]. These transients are thought to be responsible for inducing cortical granule exocytosis to prevent polyspermy, and for oocyte activation and subsequent development.
Repetitive Ca
2+
transients can also be induced artificially using various agents [2]. Among them is thimerosal (sodium ethylmercurithiosalicylate), a sulfhydryl reagent that causes Ca
2+
release in a number of cell types by modifying sulfhydryl (—SH) groups on intracellular Ca
2+
release proteins. Thimerosal has been shown to induce repetitive Ca
2+
transients in hamster [3, 4], mouse [2, 5, 6], rabbit [7], bovine [8], and human [9, 10] oocytes.
Exposure of mouse oocytes to concentrations of thimerosal adequate to stimulate a train of Ca
2+
spikes (10, 50, and 100 &mgr;M) for 5 or 30 min induced zona pellucida hardening; 39 out of 40 (98%) mouse oocytes exposed to 100 &mgr;M thimerosal for 5 min followed by a 10 to 30 min wash showed cortical granule exocytosis [5]. Although thimerosal induced cortical granule release in this system, it did not activate oocytes: no polar body extrusion was observed in either control oocytes or oocytes treated with 20-100 &mgr;M thimerosal for 30 min followed by a two hour recovery period. Furthermore, the spindle was completely destroyed in 46 out of 50 (92%) oocytes exposed to 100 &mgr;M thimerosal for 11 to 40 min, and was severely disrupted in the remaining four oocytes. Thus, treatment of oocytes with thimerosal alone under these conditions does not lead to complete activation.
An earlier study carried out on purified porcine brain tubulin indicated that the oxidation of tubulin sulfhydryl groups by 5,5′-dithio-bis(2-nitrobenzoic acid) interferes with tubulin polymerization [11]. The same study also showed that the oxidation of tubulin sulfhydryl groups could be reversed by the sulfhydryl reducing agent dithiothreitol (DTT). In the mouse system [5], 73% (30 out of 41) of mouse oocytes exposed to 100 &mgr;M thimerosal for 5 min, then 20 &mgr;M thimerosal for 20 min, followed by a wash in thimerosal-free medium containing DTT for 15 min, regenerated typical metaphase spindles. Of the remaining 11 oocytes, 10 had slightly abnormal spindles, and one lacked a spindle.
Nuclear Transfer to Oocytes
Nuclear transfer is a procedure involving the replacement of the nucleus of one cell with that of another, permitting the creation of genetically identical individuals. Recent reports on the cloning of sheep and cattle through nuclear transfer, such as the birth of “Dolly” the cloned lamb (Wilmut et al. (1997)
Nature
385:810-813), have focussed much attention on this aspect of reproductive biology. Despite the ability to clone sheep and cattle using nuclear transfer, a need exists for methods that will improve the probability of success of developing mammalian oocytes to produce embryos, fetuses, and eventually, infant and adult animals for agricultural, biomedical, and basic research purposes.
One means of cloning by nuclear transfer involves first treating an unfertilized egg with cytoskeletal inhibitors to depolymerize the microtubules and/or microfilaments, thereby imparting elasticity to the plasma membrane. The egg is then held in place with a fire-polished holding pipette, and another beveled micropipette is inserted through the zona pellucida (the protective layer surrounding the egg) of the oocyte and thrust into the cytoplasm. Since the plasma membrane is very elastic, it invaginates around the pipette. The chromosomes are located, either directly or after treatment with a DNA-specific dye and ultraviolet light illumination, and aspirated into the pipette. the pipette is then withdrawn, and the zona pellucida pinches off the cell membrane. This results in a membrane-bound nucleoplast in the pipette, and an oocyte containing no nucleus or chromosomes. A nuclear donor is then placed within the zona pellucida next to the recipient oocyte using a micropipette. The two cells are then induced to fuse using viral, chemical, or electrical stimuli to induce the fusion process. This results in deposition of the nucleus into the cytoplasm of the recipient oocyte, and a mixing of the cytoplasmic contents of the two cells. Many technical variations of this process can be employed without loss of subsequent development (reviewed by R. Prather (1996)
Proc. Soc. Exp. Biol. Med
. 212:38-43).
Next, there is a reconfiguration of the structure of the nucleus accomplished by a two-way exchange of proteins between the cytoplasm of the recipient oocyte and the chromatin of the donor nucleus. RNA synthesis is altered, the process being described as “nuclear reprogramming.” If the nucleus is truly reprogrammed, it will reinitiate its developmental pathway and recapitulate early developmental events of the embryo (Prather et al. (1991) In
Animal Applications of Research in Mammalian Development
, R. A. Pedersen et al., Eds., The Cold Spring Harbor Laboratory Press, pp. 205-232).
Thirdly, another event that must occur for subsequent development of the nuclear transfer embryo is activation of the oocyte. The mammalian oocyte is generally arrested at metaphase-II of meiosis. Fertilization through the sperm-oocyte interaction triggers an increase in the intracellular free Ca
2+
concentration ([Ca
2+
]
i
) in the oocyte, which, as noted above, takes the form of a series of repetitive Ca
2+
transients in mammals [1]. These Ca
2+
transients are thought to be responsible for inducing exocytosis of specialized secretory vesicles, described as cortical granules, which are located under the plasma membrane of the egg cytoplasm granules. The contents of the granules are thought to be responsible fo the prevention of polyspermy and for breaking the arrest of meiosis, thereby activating development of the oocyte. Additionally, other features associated with the activation of oocytes include polar body extrusion, hardening of the zona pellucida, and regeneration of the metaphase spindles, which leads to the ability of the oocyte to support subsequent development.
When performing nuclear transfer, it is necessary to artificially break this meiotic arrest, otherwise the nuclear transfer recipient will remain arrested at the 1-cell stage with condensed chromosomes. Artificial methods of activating oocytes include the electrical pulse used for fusion of the donor cell, or chemical activation.
Many methods of oocyte activation have been described for the pig. However, many procedures that work well in other species do not work well in the pig. Furthermore, many previous activation protocols only work in vivo, which involves considerable expense to perform. Thus, there is a need for methods that result in in vitro development to the blastocyst stage in porcine oocytes.
SUMMARY OF THE INVENTION
The present invention meets the foregoing need in the art by providing methods enabling either in vitro or in vivo development of activated oocytes.
As discussed above, previous studies have shown that oxidation of tubulin sulfhydryl groups can be reversed by DTT, and metaphase spindles destroyed by oocyte exposure to thimerosal can also be regenerated by exposure to DTT. No other effects of thimerosal/DTT treatment have been reported. While other workers have attempted to use thimerosal to induce activation, no observations other than a series of calcium transients, zona hardening, and cortical granule exocytosis have been reported. In
Machaty Zoltan
Prather Randall S.
Crouch Deborah
Senniger Powers Leavitt & Roedel
The Curators of the University of Missouri
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