Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal – Via microinjection of dna into an embryo – egg cell – or...
Reexamination Certificate
1999-12-28
2002-12-10
Priebe, Scott D. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
Via microinjection of dna into an embryo, egg cell, or...
C800S018000, C800S021000, C800S008000, C800S013000, C435S455000
Reexamination Certificate
active
06492575
ABSTRACT:
The invention relates to the production of mammals with defined genetic properties, particularly the production of transgenic animals.
Transgenic animals are organisms into whose germline permanent genetic changes have been introduced; a newly introduced gene is known as a transgene. Transgenic animals constitute an essential tool in modern biology for analysing the tissue-specific regulation of genes and their function in development and in diseases. Moreover, transgenic technology provides an opportunity of having animal models available for diseases in humans and producing large amounts of proteins in farm animals.
In the method of producing transgenic animals which has hitherto been used most frequently, recombinant DNA is microinjected into the fertilized eggs; another technique for introducing genes into animal embryos makes use of viruses, usually recombinant retroviral vectors (cf. the summarising articles by Wagner and Keller, 1992).
The third and most recent technique for introducing foreign genetic material into animals makes use of the potential of embryonic stem cells (ES cells) to create chimeric animals. Mammalian embryos have the capacity to incorporate foreign cells during their development. Two different pre-implantation embryos, usually morulae, are aggregated in vitro; this produces a chimeric embryo, which constitutes a mixture of the two embryos. These embryos are then transferred into a pseudo-pregnant mouse which acts as a foster mother; the chimeric offspring obtained have, in their tissues, different numbers of cells which originate from one of the two original embryos. Combining this method with the use of ES cells has proved very effective in the production of genetically manipulated animals.
Embryonic stem cells are derived from the inner cell mass (ICM) of blastocysts; they are totipotent cells which are capable of developing into all cell lineages, including germ cells, when introduced into an embryo by injection into diploid blastocysts or by aggregation with morulae (Robertson, 1987; Bradley, 1987; Beddington and Robertson, 1989; Nagy et al., 1990). ES cells can be isolated from blastocysts and then established as permanent cell lines if they are cultivated under well defined culture conditions which are strictly adhered to; they can be genetically manipulated. In view of this ability, they constitute an effective tool for modifying the mammalian and particularly the mouse genome by being introduced into the animals, for example, by means of controlled mutations or other genetic modifications (Wagner et al., 1991; Ramirez-Solis et al., 1993; Skarnes, 1993; Bronson and Smithies, 1994).
For some time, cells designated “embryonic germ cells” (EG cells) have been available, which can be cultivated from primordial germ cells into immortalised cell lines and are similar to ES cells in many respects; EG cells are, inter alia, totipotent, can be manipulated in the same way as ES cells and form germline chimeras when introduced into blastocysts (Donovan et al., 1997).
In recent years, various experimental techniques have been developed for producing animals derived from totipotent cells. (Totipotent cells are cells with the ability to differentiate themselves into all somatic cells as well as germ cells.) In the case of ES cells the primary objective of these methods was to obtain the entire developmental potential of ES cells in vitro (Williams et al., 1988; Smith et al., .1988) and to restrict the developmental potential of the host cells in the formation of chimeras and thus increase the frequency of forming germline chimeras (Nagy et al., 1990; Kaufman and Webb, 1990). One of the most important pieces of progress in the development of these techniques is the use of tetraploid embryos as host cells, because tetraploid cells have only restricted potential for development after they have been implanted (Nagy et al., 1990; Kaufmann and Webb, 1990; Kubiak and Tarkowski, 1985). When tetraploid embryos are aggregated with diploid embryos, the differentiation of the tetraploid cells is largely restricted to the primitive endoderm and the trophectoderm, which subsequently form extraembryonic tissue, whereas the diploid cells can form the actual embryo (James and West, 1994; James et al., 1995).
In an earlier study, various ES cell lines were aggregated with morulae in order to produce fetuses which are derived completely from ES cells (organisms derived completely from ES cells are hereinafter referred to as ES animals, e.g. ES mice or ES fetuses; however, the ES foetuses obtained died at birth (Nagy, 1990). Further studies showed that viable, fertile ES mice derived exclusively from ES cells can be obtained if wild-type R1 cells of an earlier passage (Nagy et al., 1993) or TT2 cell lines (Ueda et al., 1995) are used for the aggregation with tetraploid morulae.
Moreover, ES mice are produced by injecting ES cells into diploid blastocysts in a first step, thereby initially obtaining chimeric mice; further crosses produced ES mice after two generations. The method of injecting into blastocysts was first described by Gardner, 1968, and a simplified version was described by Bradley and Robertson, 1986, and by Bradley, 1987.
The objective of obtaining viable ES mice by using ES cells from later passages has not been achieved with the methods available hitherto (Nagy et al., 1993); it did not seem possible to produce ES mice at all using genetically modified ES cells. (The possibility of using ES cells from later passages is significant particularly in view of the use of cell lines and also with respect to the use of genetically modified ES cells the selection of which naturally goes hand in hand with an increase in the number of passages.)
The aim of the present invention was to provide a new process by which mammals with defined genetic properties, particularly transgenic mammals, can be obtained which are derived completely from totipotent cells.
The objective is achieved by means of a process for producing mammals with defined genetic properties, particularly transgenic mammals, wherein totipotent cells of the same mammalian species are introduced into blastocysts and the resulting embryo is transferred into a foster mother. The process is characterised in that totipotent cells with defined genetic properties are introduced into tetraploid blastocysts.
Using the process according to the invention it is possible to obtain animals which are completely derived from totipotent cells. The process according to the invention has the advantage that animals derived totally from totipotent cells are obtained in a single step from totipotent cells cultivated in vitro (ES cells or EG cells).
The term “transgenic mammals” includes, for the purposes of the present invention, animals which have a permanent genetic modification of any kind.
Animals which “are totally derived from totipotent cells” preferably contain up to 100% of cells originating from ES cells or EG cells. However, the animals may contain a small proportion, preferably not more than 10%, of cells derived from the tetraploid blastocysts.
In a preferred embodiment of the invention the mammals are mice; however, the process may also theoretically be applied to all mammals from which ES cells or EG cells can be obtained. The prerequisite for obtaining totipotent cells from mammals other than mice is the definition of conditions which allow the cultivation of ES cells or primordial germ cells from these organisms and the establishing of ES or EG cell lines, which include, inter alia, the need for specific growth factors as well as feeder cells for co-cultivation with the ES cells or EG cells. These conditions can be determined empirically by series of tests.
The isolation of ES cells from blastocysts, the establishing of ES cell lines and their subsequent cultivation are carried out by conventional methods as described, for example, by Doetchmann et al., 1985; Li et al., 1992; Robertson, 1987; Bradley, 1987; Wurst and Joyner, 1993; Hogen et al., 1994; Wang et al., 1992. The cultivation of EG cells can be carried
Wagner Erwin
Wang Zhao-Qi
Boehringer Ingelheim International GmbH
Paras, Jr. Peter
Priebe Scott D.
Sterne Kessler Goldstein & Fox P.L.L.C.
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