Production Of Chimeras Research Articles

Production of ovine chimeras by inner cell mass transplantation.

Ovine chimeras were produced by micro-injection of isolated inner cell masses (ICM) into recipient blastocysts. Inner cell masses were isolated by immunosurgery. A total of 57 chimeric embryos was produced, 52 of which were transferred to recipient ewes. Thirty-seven live lambs were born, of which 15 were determined to be chimeric on the basis of blood type analysis. One lamb, although not a blood chimera, exhibited overt signs of chimerism. An additional six lambs were determined to have developed solely from the injected ICM. The rate of chimerism in live lambs was 43% (16/37) while the survival rate of injected ICM was 59% (22/37). The method presented allows the production of relatively large proportion of viable, chimeric embryos without the use of an intermediate recipient.

Successful rescue of microsurgically produced homozygous uniparental mouse embryos via production of aggregation chimeras.

Homozygous uniparental mouse embryos, produced by microsurgical removal of the male pronucleus from fertilized eggs and diploidization of the female pronucleus with cytochalasin, were surrounded with blastomeres from normal embryos to produce chimeric embryos. A few of these chimeras developed into viable adults, and one female has reproduced using her homozygous uniparental cells as a source of gametes. The production and use of such chimeras in breeding programs could greatly shorten the period required for producing inbred strains of mammals. The data presented demonstrate that a homozygous uniparental mammalian genome, although not lethal to all cells, is extremely deleterious to normal embryonic development. Moreover, the results support the conclusion that the genome is imprinted differently in males and females during gametogenesis so that at fertilization the genomes are not functionally equivalent, and both are required for normal development.

Production of experimental chimeras in livestock by blastocyst injection.

Mammalian embryos have the interesting property that allows them to be combined to form a normal individual containing cells of 2 distinct geno-types. Although this mixing of embryonic cells or chimerism occurs naturally (see Ref. 3 for review), whole body chimerism is relatively rare. Nicholas and Hall (13) attempted to produce chimeras in the laboratory by aggregating one-cell rat embryos, however, no conclusive evidence could be offered in terms of chimerism. Tarkowski (16) was the first to develop a procedure by which chimeras could be readily produced. Immediately following, Mintz (10,11) published a similar method but with modifications that resulted in a simpler procedure. Briefly, the zonae pellucidae were removed either mechanically or enzymatically from early cleavage-stage embryos, the 8-cell stage being the most common. Two embryos of different strains were then placed in contact with each other and allowed to aggregate into a single embryo. The chimeric embryos were allowed to develop to the blastocyst stage and then transferred to suitable recipients. The ability to readily produce large numbers of whole body chimeras proved to be a valuable tool in the study of embryology and developmental genetics (see Ref. 9 for review).

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.

Experimentally Synthesized Plant Chimeras 2. A Comparison of In vitro and In vivo Techniques for the Production of Interspecific Nicotiana Chimeras*

In vitro and in vivo techniques were compared for synthesizing chimeras between Nicotiana glauca Grahm and N tabacum L Interspecific chimeral callus, produced from mixed callus cultures in vitro, was placed on media which favoured only N tabacum shoot formation None of the 474 regenerated N tabacum shoots incorporated N glauca cells into their meristems When chimeral callus was regenerated under hormonal conditions favouring simultaneous organogenesis, of 397 shoots, only non-chimeral shoots of both species arose In vivo, reciprocal splice grafts between species were decapitated just above the graft union and treated with or without auxin—lanolin pastes Auxin increased callus formation but inhibited adventitious shoot formation Three of 209 adventitious shoots arising from the graft union were interspecific mericlinal chimeras which were later stabilized as periclinal chimeras All three chimeras formed when N glauca was the understock Two of the chimeras arose on untreated shoots which produced no visible callus, indicating that excessive callus formation may be unnecessary for multiple cell origin of adventitious shoots to occur.

Purification and properties of chick terminal deoxynucleotidyl transferase (TdT).

The thymus is made up of two anatomical parts: the stroma, containing the epithelium, and the thymocytes which belong to the lymphoid series of the hematopoietic cells. The embryonic origins of these two populations are different: the thymocytes derive from stem cells which colonize the thymic rudiment (which is initially purely epithelial). This facts were suggested by Moore and Owen (1) and the timing of the lymphoid colonization of the embryonic thymus was established by Le Douarin and Jotereau (2) using a technique based on the production of interspecific chimeras. Such chimeras are obtained by grafting the thymic rudiments of chick embryos in the somatopleure of quail embryos (or vice-versa). Quail cells are easily distinguished from chick cells using the Feulgen-Rossenbeck staining of chromatin: the quail chromatin is condensed in a large mass in the center of the nucleus whereas the chick chromatin is finely dispersed (3). By an appropriate choice of the respective ages of the grafted rudiments and of the host embryos, it could be shown that the epithelial thymus is colonized by stem cells during two receptive periods : from 6 1/2 days to 8 days and from 12 to 13 days.KeywordsChick EmbryoTerminal DEOXYNUCLEOTIDYL TransferaseEpithelial ThymusQuail EmbryoReceptive PeriodThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Production of chimeras in the deer mouse Peromyscus maniculatus bairdi

AbstractThe production of intraspecific chimeras in the deer mouse, Peromyscus maniculatus, was undertaken to make mammals of another genus useful for studies of reproductive biology, developmental genetics, and embryonic development. Peromyscus was generally amenable to the same experimental procedures and techniques employed in producing Mus musculus chimeras. We found that Peromyscus embryos could be successfully manipulated and cultred in vitro. Fertilized eggs and 2‐cell embryos continue cleavage when cultured in vitro and form morulae and blastocysts. Almost all the 4‐ and 8‐cell embryos that were aggregated remained together and developed, after 1 day of culture in vitro, to form chimeric morulae and blastocysts. However, certain changes had to be made in the chimera‐making procedure employed for M. musculus to accomodate some of the differences in reproductive physiology and behavior of Peromyscus. The methods for the preparation of donor females and of foster mothers had to be modified in order to obtain embryos on the scheduled day of the experiment, and to obtain pseudopregnant foster mothers. With these revised precedures, three P. m. bairdi chimeras were produced. Two are healthy fertile males displaying chimerism in the fur. The other chimera was delivered dead at 17 days of gestation, after its foster mother died. Chimerism was detected in the retinal epithelium of the eye by the presence of black pigmented patches contributed by the wild‐type genotype, and unpigmented patches, contributed by the blonde mutant genotype. By developing the procedures enabling the production of the first chimeras in this genus, we have demonstrated that Peromyscus is a suitable animal for exacting technical manipulations in the laboratory.

Changes in the properties of the developing trophoblast of preimplantation mouse embryos as revealed by aggregation studies

ABSTRACT Mouse embryos (8-cell to early blastocyst) were denuded with pronase, and apposed in pairs which represented a wide range of stage combinations. These pairs either formed aggregates which differentiated into double-sized blastocysts, or they failed to aggregate. The 8-16-cell stages would not envelop late morulae/early blastocysts to form layered aggregates. This must mean that as the embryo differentiates into a blastocyst, the outer surface of the trophoblast loses its capacity for supporting cell spreading. The aggregation data also demonstrate that embryos almost completely lose their potential for aggregation at a very discrete stage in development -namely, between 8 and 9 h before blastocoel formation. It is argued that this is the stage at which the zonular tight junctional seal is completed, and that it is this physical barrier which prevents aggregation. It has been argued previously that the zonular tight junctional seal allows the creation of the special microenvironment which is necessary for the determination of the inner cells as inner cell mass. The completion of this seal 8-9 h before it is required for the formation of a blastocoel would provide a suitable time period for this cell determination to occur. The results obtained also relate to the technique of chimera production. Since the aim of this technique is to generate mice with mixed cell populations, it is important that the blastocyst formed following aggregation should have both cell lines present in the inner cell mass. This can best be assured by using relatively late morula stages (75 h post-HCG injection) since these will have already segregated their inner cells, but the incomplete seal will still allow aggregation to take place.

Production and Metamorphosis of Chimeras in Anurans and Urodeles

The perfect union of the anterior and posterior halves of the embryos of 2 species of anurans and the metamorphosis of such a combination into a perfect adult was shown to be possible by Harrison 30 years ago. Up to the present day the opportunities which this operation makes possible have never been realized. The intention of the present report is to demonstrate that such combinations can be produced in large enough numbers to enable the experimental embryologist and geneticist to carry out a variety of new studies. The frog embryos employed were Rana palustris and Rana sylvatica. Those of the salamander used were Amblystoma tigrinum and Amblystoma punctatum. The stages at which the operation was done were in the early tail-bud period of development. The species conbinations in their respective classes are, at the tail-bud stages, about the same in size. Similar stages in development were selected and placed in slightly diluted Ringer's solution in an operating dish, the floor of which was covered by a thick film of white beeswax. The embryo was oriented under the microscope, dorsal side up, between fine forceps so that iridectomy scissors could, with one stroke, cut the embryo transversely at the level of the seventh to eighth somites. It is quite possible to cut at other levels as well in making successful combinations. The anterior half of one species and the posterior half of the other were then placed in a depression in the wax which was about the size of an intact embryo. The cut surfaces of the reciprocal halves were brought together in the concavity of the wax as soon after the operation as possible.