Method to produce a hybrid cell having a single mitochondrial genotype

#The present invention relates in at least some of its aspects to methods of producing cells, and to improved cloning methods, as well as to cells and non-human animals produced by said methods.

#The technique of nuclear transfer (NT) allows the production of offspring by the reconstruction of an oocyte, egg, zygote, or early embryo. Genetic material from a donor cell (sometimes referred to as a karyoplast) is transferred to a suitable recipient cell (sometimes referred to as a cytoplast) from which the nuclear or genomic genetic material has been removed. In the first demonstrations of this technique successful development was only obtained when the donor genetic material was taken from apparently undifferentiated cells or blastomeres from early embryos. Subsequently, donor genetic material from differentiated cells maintained in culture and isolated from embryonic (Campbell et al. Nature 380:64-66), foetal and adult tissues (Wilmut et al Nature 385: 810-813) has been successfully used. The aforementioned reports form the basis of patent applications WO 97/07669 and WO 97/07668 the contents of which are incorporated herein by reference. Subsequently, live offspring have been obtained utilising a range of cell types and techniques e.g. in mouse using cell populations derived directly ex vivo as nuclear donors (Wakayama et al., Nature 394:369-373) and from a range of cultured cell types including foetal fibroblasts. More recently live piglets were produced using a modified NT procedure (Polejaeva et al., Nature 407: 29-30, WO 00/22098). The contents of the aforementioned publication and patent application are incorporated herein by reference. The successful use of differentiated cells has now been demonstrated in sheep, cattle, goats, pigs and mice.

#The use of nuclear transfer technology has many benefits and uses in the production of mammalian embryos foetuses and offspring. These include but are not limited to; 1. The ability to carry out precise genetic modification of cultured cells to be used as nuclear donors prior to embryo reconstruction. Such modifications include but are not limited to, random gene addition, addition of multiple copies of a transgene, addition of a transgene at a precise location (targeted addition or “knockin”), gene removal (“knockout”), gene inactivation by targeted insertion, gene replacement, and modification of any gene or its control sequences and gene multiplication. 2. The ability to carry out multiple genetic modifications in a single animal either by multiple genetic modifications of a cell population in culture or by sequential genetic modification, nuclear transfer and re-isolation of a cell population from the embryo, foetus or animal so produced. 3. The ability to increase the lifespan of cultured cell populations to be used for genetic modification by nuclear transfer and re-isolation of a cell population from the embryo, foetus, juvenile or adult animal so produced. 4. The ability to produce multiple copies of an animal from a genetically modified selected and cloned cell population. 5. The ability to produce multiple copies of any embryo, foetus, juvenile or adult animal by nuclear transfer from cells taken directly ex vivo, or cell populations derived from any tissues taken from any of these stages with or without culture in vitro. 6. The ability to store intact genomes for long periods (e.g. by freezing cell populations in liquid N2) and to subsequently use these stored cells for the production of offspring by nuclear transfer. 7. The ability to dedifferentiate somatic nuclei and to produce undifferentiated cells that may be used for production of chimeric embryos, foetuses and adult animals by embryo aggregation or injection or to produce embryonic stem cell or embryonic germ cell populations. 8. The ability to dedifferentiate any somatic cell type by nuclear transfer and to isolate from the embryo so produced embryonic stem cells, germ cells or any other desired specialised or unspecialised cell type e.g. neurones.

#1. The Generation of Primary Cultures of MtDNA Depleted Ovine Foetal Fibroblast Populations.

#Primary cultures of ovine foetal fibroblasts were isolated from day 30 foetuses as described by Schnieke et al. (1997) (Science 1997; 278: 2130-2133). Isolated cell populations were cultured in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 (1:1v/v) supplemented with 10% FCS (v/v), 15 mM HEPES, 1.2 g/litre sodium bicarbonate, penicillin and streptomycin, pH7.4. Fibroblasts were depleted of mitochondria or mtDNA according to the protocol of King & Attardi (supra). Briefly, fibroblasts were cultured in DMEM containing 4500 mg/litre glucose and 1 mM pyruvate and supplemented with 5% FBS (v/v), 100 μg bromodeoxyuridine/ml and 50 μg/ml uridine. Cells were grown in the presence of, for example ethidium bromide or Rhodamine 6-G, in order to isolate cells completely lacking mtDNA or having dysfunctional mtDNA or completely lacking mitochondria. After mitochondrial depletion, the cells were maintained in DMEM supplemented with 5% FBS, 100 μg bromodeoxyuridine/ml and 50 μg/ml uridine. In order to determine that the isolated cells are mitochondrially depleted, a batch of the cells were cultured in an aerobic environment, i.e. DMEM supplemented with 5% dialysed FBS and 100 μg bromodeoxyuridine/ml. The absence of a pyrimidine (uridine, in this instance) source in this selection media ensured that those cells unable to generate ATP through OXPHOS would not survive.

#2.1. Superstimulation of Donor Ewes and Recovery of Oocytes.