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
1999-10-12
2003-07-29
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...
C800S008000, C800S015000
Reexamination Certificate
active
06600087
ABSTRACT:
The present invention relates to a process for breeding animals through cloning as well as the animals that can be obtained with the process. This relation relates in particular to a process for cloning animals by an efficient nucleus transfer with specific foetal cells.
Animals, in particular working animals, have been bred by humans for a long time for many different purposes, and developed with regard to particular characteristics. Thus, for example, cows and bulls with a high breeding value for milk yield, were mated in order to obtain animals with a high milking capacity.
In recent years, animals, in particular representatives of the ungulates, such as sheep, cattle and cows have also become the focal point of research interest as production centres of material that are important in terms of nutrition physiology and pharmaceutics, as with the development of genetic engineering it became possible to manufacture in a targeted fashion animals to which a new property, for example, the ability to produce a specific drug, could be given. The problem with the commercial exploitation of such animals, however, is that the genetic construct transferred to them is passed on to their descendants in an integrated and stable manner.
To this end, attempts were made to solve the problem of gene transfer by carrying it out in cells, animals then again being generated from these cells by means of cloning.
Among experts, the term “cloning” is defined generally as the multiplication of a genetic material derived from one single cell, which, transferred to embryology, can be understood as the creation of embryos or animals with an identical genotype. In embryology, the fertilized egg is described as an embryo during blastogenesis, that is, up to the development of the primordium of primitive organs, and as a foetus in the subsequent development stages. Embryonic phases last for different periods of time in different species, thus for example in cattle for a period of about 4 weeks, while shorter or longer periods of time may be required for this for other species within the ungulates.
Up to now, several routes have been followed for cloning animals, that is, for the multiplication of a genotype specific to a particular animal.
On the one hand, early embryonic stages and developments were subjected to microsurgery and the parts isolated from each were bred in vitro or in vivo respectively.
Furthermore, a micromanipulatory combination of asynchronous development stages called “chimeric cloning was carried out in which blastomers from embryos of more advanced stages were brought together with blastomers from earlier stages with the aim of supporting the former in their further development capacity and thus produce identical multiple twins. The largest number of clones obtained in this way, however, was only of the order of 5-8 maximum.
Another procedure was the parthenogenetic activation or mating of homozygous parent animals to obtain clones with regard to specific properties.
However, as the processes named above proved to be relatively poor in terms of effectiveness and reliability, a further process was developed which is generally described as nucleus transfer.
In this process, cell nuclei, which come from multicellular embryos are transferred into suitably prepared egg cells, genetically identical embryos being able to be created.
To be able to carry out a cloning successfully by means of nucleus transfer, however, some in dispensible parameters must be taken into account.
The egg cell that is used as a receptor cell must have completed the metaphase stage in the second division of maturation (metaphase II) and is to no longer contain nucleus DNA of its own, i.e., it is to be present as a so-called enucleated egg cell. Furthermore, the egg cell cytoplasma should be influenced as little as possible, as the substances contained in the cytoplasma itself can be significant for development, for example, the division of the cell.
In addition, the nucleus DNA of the transferred nucleus must be reprogrammed. As the (donor) nucleus comes from a multicellular embryo, the respective donor cell has already passed through some division cycles. This means that the cell is in a development stage which is advanced compared with a totipotent fertilized egg cell in which certain genes which are required for early development are possibly already switched off.
For this reason, the nucleus DNA used must be reprogrammed in such a way that the complete genetic information of the nucleus DNA is available again and the division programme of the embryo begins again at the zygote stage. The better this reprogramming or activation can thus be achieved, the greater the possibility of a successful cloning with which a ready developed cloned animal, i.e. one born alive can then also be obtained.
In addition to the nucleus DNA, the mRNA also present in the cytoplasma is also significant, inter alia, as at the time of the unification of egg cell and donor cell, this represents the messages required for the current development or differentiation stages of the donor cell and the proteins manufactured in this way can have an influence on the further development of the cell.
The process of nucleus transfer has already been used with modest success. Thus, Willadsen et al. reported (Nature 320 (1986), 63-65) on the cloning of lambs, the nuclei coming from nucleus donor cells from the 8-cell stage. Robl et al. (J. Anim. Sci. 64 (1987), 642-647) reported on the first nucleus transfer experiments in cattle, cattle embryos obtained ex vivo being exclusively used as nucleus donors. In these tests, an in vivo intermediate culture in sheep fallopian tubes was always required. In the following years, it was also shown that embryo cloning in cattle can be carried out successfully purely in vitro, i.e. using embryos produced in vitro and egg cells matured in vitro (Sims et al., Proc. Natl. Acad. Sci. USA 91 (1991), 6143-6147).
In WO 97/07668, a process is furthermore described for the reproduction of a an animal embryo in which generally a nucleus with a diploid chromosomal set is transferred to an enucleated egg cell which is maintained in the metaphase stage II, the egg cell first being activated when the nucleus is introduced only after a specific time. By activating the egg cell later after the introduction of the nucleus DNA, an improved reprogramming of the introduced nucleus DNA used is to be achieved.
WO 97/0669 also relates to a process for the reproduction of an animal embryo in which the nucleus of a “quiescent” (dormant) donor cell is transferred into a suitable receptor cell. According to this publication, it is regarded as necessary that the donor cell is fixed in the GO phase before unification with the receptor cell, which can be achieved by starving the cells or by contact inhibition.
One problem with this technology, however, is still to find suitable donor cells for nucleus transfer with which an animal embryo can most expediently and economically be produced. As is known, reprogramming the nucleus DNA from the respective donor cell chosen presents the greatest difficulty when cloning embryos as this not only influences the further early maturation of the embryos, but also the later development after an implantation into a mother animal that is possibly carried out. Thus, despite all the successes in this area, there are still problems with regard to an effective reprogramming of the donor-nucleus-DNA in order to bring the manipulated egg cell with the new nucleus close to the state of a natural zygote. This is manifested, inter alia, in an extremely small yield with regard to the production of embryonic blastocysts and a low division rate.
One object of the present invention is therefore to overcome the disadvantages of the state of the art and to provide a suitable nucleus-donor cell with which an improved process for cloning animals can be provided.
This object is achieved by a process for cloning an animal embryo in which foetal fibroblasts are used as a donor cell for the nucleus transfer. The nucleus of this ce
Brem Gottfried
Durcova-Hills Gabriela
Müller Sigrid
Schernthaner Wolfgang
Wenigerkind Hendrik
Agrobiogen GmbH
Crouch Deborah
Katten Muchin Zavis & Rosenman
LandOfFree
Efficient nuclear transfer using fetal fibroblasts and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Efficient nuclear transfer using fetal fibroblasts and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Efficient nuclear transfer using fetal fibroblasts and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3031454