Genetic Copies Research Articles

Donor Cells for Nuclear Cloning: Many Are Called, but Few Are Chosen

The few viable clones obtained at the end of a typical cloning experiment are genetic copies of the donor cell genome of a non-reproductive (somatic) or embryonic cell used for nuclear transfer. Nuclear totipotency has to be reestablished by erasing epigenetic constraints imposed on the donor genome during differentiation in a process which involves active chromatin remodeling. Various donor cell types and cell cycle combinations have proven to be capable of generating cloned offspring. However, an ideal nuclear donor may have not yet been found. This review summarizes current theoretical aspects of donor cell selection. It focuses on the impact of genetic and epigenetic differences between donor cell types on successful mammalian cloning.

Cloning, transgenesis, and genetic variance in animals.

251 THIS SYMPOSIUM, “Methods for Manipulating the Embryonic Genome In Vitro,” provides much material for further discussion. Simply to review these reviews would constitute failure to learn from the observation that there are nearly as many reviews of transgenesis as original papers (Wall, 2001). Paradoxes abound when considering the subject matter of the symposium; one example is that one of the most important tools for transgenesis, which can increase genetic variance, will likely be cloning by nuclear transplantation/cell fusion, which has the reputation of decreasing genetic variance. Another paradox is that genetic knockouts produced by transgenic techniques are the ultimate in decreasing genetic variance, resulting in deleting the function of an entire gene. To the knowledgeable scientist, the papers in this symposium constitute a collection of technologies for making genetic modifications in germ-line cells (Seidel, 2000). Cloning by nuclear transfer, as suggested above, can serve as a mechanism for moving genetically modified embryonic or somatic genomes into the germ-line. The seminal paper by Wilmut et al. (1997) on producing offspring via nuclear transfer using cells derived from somatic fetal fibroblasts as nuclear donors provided strong evidence that this would be possible; subsequent publications have substantiated this supposition. The realization that it is possible, albeit with relatively low efficiency, to move genetic material from somatic cells to germ-line cells constitutes a paradigm shift, much as occurred when the central dogma of molecular biology (DNA RNA protein) was shaken when the discovery of reverse transcriptase demonstrated that the first arrow was reversible. In the same way as reverse transcriptase has become an everyday tool in molecular biology, cloning animals from somatic cells is becoming a laboratory resource for researching other questions rather than just an end in itself. This is illustrated in the paper by Campbell et al. (2001), which describes various possible applications of cloning, including rescuing cell lines from senescence (Denning et al., 2001). This recovery mechanism that resets cellular youth is yet to be characterized, and it may or may not be as simple as regulation of telomerase genes (Oback and Wells, 2002). If electronic databases of biological research are searched using the keyword “cloning,” one comes up with many more papers on cloning DNA than making genetic copies of living organisms, and only a small proportion of the latter concern mammals. But cloning DNA illustrates a point nicely: for most rational transgenic experiments, despite recent advances (e.g., Maga, 2001), millions of identical copies of the transgenic construct are required. Similarly, for genetic manipulation of populations of animals, more than one genetic copy often will be useful, particularly if the copies can be of different ages. Several concepts of genetic variability will be described briefly. There are many ways of thinking about genetic variance, some of which are listed in Table 1 for farm animals (although most of these concepts apply to any species). In practical terms, genetic variance often is most relevant at the level of the herd or flock, since that is the economic unit being managed. The success of the herd, of course, depends on individual animals

Donor Cells for Cloning—Many Are Called But Few Are Chosen

The few viable clones obtained at the end of a typical cloning experiment are genetic copies of the donor cell genome of a nonreproductive (somatic) or embryonic cell used for nuclear transfer. Nuc...

The future of cloning.

It is now possible to make clones, or exact genetic copies, of sheep, cows, goats, mice and, probably, humans. This opens the way towards the production of replacement body parts from adult cells.

What can we learn from natural apomicts?

Apomixis is an asexual breeding system in plants that allows fertile seeds to develop in the absence of fertilization. Apomicts produce progeny that are genetic copies of themselves, thus preserving the maternal genotype. Ever since its discovery 1 Smith J. Notice of a plant which produces seeds without any apparent action of pollen. Trans. Linnean Soc. 1841; 18: 509 Crossref Google Scholar in 1841, apomixis has been regarded as a rare and eccentric breeding system in angiosperms, but there has been much recent interest in apomixis because of its great potential for crop improvement 2 Vielle-Calzada J.P. Crane C.F. Stelly D.M. Apomixis: the asexual revolution. Science. 1996; 274: 1322-1323 Crossref Scopus (109) Google Scholar , 3 Grossniklaus U. Koltunow A. van Lookeren Campagne M. A bright future for apomixis. Trends Plant Sci. 1998; 3: 415-416 Abstract Full Text Full Text PDF Scopus (41) Google Scholar and the possibility of changing sexual crops into apomicts 4 Koltunov A.M. Bicknell R.A. Chaudhury A.M. Apomixis: molecular strategies for the generation of genetically identical seeds without fertilization. Plant Physiol. 1995; 108: 1345-1352 PubMed Google Scholar via ‘apomixis technology’. In many sexual crops, hybrid varieties are superior, but because sexual hybrids segregate, they have to be renewed each generation by crossing the parental lines. In contrast, apomictic hybrids would breed true because meiosis and segregation are by-passed. Although apomixis does not naturally occur in today's main crop species (and attempts to introgress apomixis into crops by interspecific hybridization with wild apomictic relatives have so far been unsatisfactory), modern molecular techniques are expected to give better prospects for introducing apomixis into crops. The potential applications of ‘apomixis technology’ go far beyond classical apomixis introgression, where species barriers and different ploidy levels usually prevented successful transfer of apomixis from wild plants to cultivated relatives. If a sexual plant could be transformed into an apomict by introducing an ‘apomixis cassette’, every superior multigenic genotype could be preserved and propagated rapidly as a cultivar.

Mapping diplosporous apomixis in tetraploid Tripsacum: one gene or several genes?

Polyploids in Tripsacum, a wild relative of maize, reproduce through the diplosporous type of apomixis, an asexual mode of reproduction through seeds. Diplosporous apomixis involves both the failure of meiosis and the parthenogenetic development of the unreduced gametes, resulting in progenies that are exact genetic copies of the mother plant. Apomixis is believed to be controlled by one single dominant allele, responsible for the whole developmental process. Construction of a linkage map for the chromosome controlling diplosporous apomixis in Tripsacum was carried out in both tetraploid-apomictic and diploid-sexual Tripsacum species using maize restriction fragment length polymorphism (RFLP) probes. A high level of collinearity was observed between the Tripsacum chromosome carrying the control of apomixis and a duplicated segment in the maize genome. In the apomictic tetraploid, there was a strong restriction to recombination, as compared to the corresponding genomic segment in sexual plants and maize. This suggests that apomixis, although inherited as a single Mendelian allele, might really be controlled by a cluster of linked loci. The analysis also revealed the tetrasomic nature of the inheritance of the chromosomal segment controlling apomixis, which contradicts the usually accepted hypothesis of an allopolyploid origin of apomictic species. The implications of these data for the transfer of apomixis into cultivated crops are discussed, and a new approach to studying the genetics of apomixis, based on comparative mapping, is proposed.

Manipulation of the mammalian embryo.

Technological advances in manipulation of mammalian embryos outside the maternal environment have resulted in opportunities for study of preimplantation embryo development, identification of developmental phenomena that are unique to mammals, and further improvement of technology. Mammalian embryos may be cultured in vitro at 37 C for up to several days or they may be stored at -196 C indefinitely. The mammalian embryo possesses the unique capacity to regulate its development and differentiate into a normal individual after being stimulated to incorporate foreign cells or after a portion of its cells are removed. This regulatory ability has proven useful in research dealing with the production of chimeras. It allows genetic copies of an embryo to be produced by dissociation of an early cleavage-stage embryo into its component blastomeres or by bisection of a morula. Production of large sets of identical animals may be possible in the future by serial transplantation of nuclei from one embryo into enucleated ova. Progress has been made in producing unique genetic combinations by manipulation of the pronuclei of fertilized ova. It is also possible in some cases to identify the sex of a living cleavage-stage embryo. Some of these manipulations have been carried out primarily in laboratory mice, but as animal scientists identify beneficial uses in farm animals, these procedures are being extended to embryos of the large domestic species.

Production of genetically identical sets of mammals: cloning?

In the past few years, there has been phenomenal progress in producing genetically identical mammalian multiplets by asexual means. The simplest method is to divide embryos into two, three, or four parts, and transfer the parts to surrogate mothers for gestation to term. Another approach is to inject nuclei from totipotent embryonic cells into fertilized one-cell embryos and to remove both male and female pronuclei. When these embryos reach the blastocyst stage, nuclei from their totipotent cells can be removed and injected into one-cell embryos, thus amplifying the process ad infinitum. Subsets of these embryos can be stored in liquid nitrogen to make genetic material for additional copies available indefinitely. Some of these techniques are fairly efficacious, others are not. No reliable techniques are available for making genetic copies of nonhomozygous adult mammals (or other vertebrates) unless samples of embryonic cells were cryopreserved when the individuals were embryos. However, injection of nuclei of spermatogonia into enucleated one-cell embryos is a promising strategy. The genetic totipotency of these nuclei is obvious since they become gamete nuclei. Moreover, the birth of mice from the diploidized genetic material of a single X-bearing spermatozoan after removal of the female pronucleus from a one-cell fertilized embryo suggests that we should eventually be able to make genetic copies of adult male mammals. This approach would not work for adult females because their oogonia have all become oocytes, and the DNA configuration in oocytes is not identical to somatic cells due to meiotic crossing over.