3585The most rigorous test possible for developmental totipotency would be significant contributions of the carcinoma cells to the normal differentiation of virtually all tissues in a mouse. For this to occur, the initially malignant cells would presumably have to be brought into association with early embryo cells so that the latter could provide an organizational framework appropriate for normal development. The experiment is analogous to production of allophenic mice (6).The projected teratoma studies in this laboratory involve the use of mutagenized embryonal carcinoma cells for experimental analyses of genetic regulatory systems in mammalian differentiation (7). The most promising source of baseline, or nonmutagenized, cells appears to be from the cores of small embryoid bodies grown only in vivo, as these would be less likely to have accumulated genetic changes than would cells cultivated in vitro. Most in vitro teratocarcinoma cell lines do not in fact have a normal chromosome number and some have lost multipotentiality (8-11), whereas the tumors from which they originated were diploid (12).We report here that teratocarcinoma cells taken from the cores of embryoid bodies grown only in vivo for 8 years, during which they retained a euploid chromosome complement, appear to be developmentally totipotent. The original conversion to malignancy in all likelihood did not involve mutational events and has proved to be completely reversible to normalcy. A partial report of these results has been presented (7). MATERIALS AND METHODSCore Cells. The OTT 6050 ascites teratoma, originally received from Dr. L. C. Stevens, has been maintained by intraperitoneal transfers every 2-3 weeks in syngeneic males of the 129/Sv SlJ C P inbred strain (to be referred to as 129) (see Fig. 1). To obtain core cells, only small-size embryoid bodies were collected, in Dulbecco's phosphate-buffered saline (PBS), by filtration of ascites fluid through 100 /Am Nitex mesh (13). They were subjected to light proteolytic treatment for approximately 3 min in 0.25% trypsin (crystalline, Worthington) and 1.25% pancreatin (Difco) in PBS; proteolysis was stopped in Dulbecco's modification of Eagle's medium (DMEM) with 50% fetal calf serum (FCS) (Gibco). Addition of 5 ,gg/ml of DNase eliminated stickiness.After about 15 min, the embryoid bodies were centrifuged and transferred to DMEM with 15% FCS and organic buffers [10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) and 10 mM morpholinopropane sulfonic acid (Mops)], and the epithelium was peeled off with tungsten needles. The cores (Fig. 3) were then partially dissociat- [+/+].) The embryo was placed under a testis capsule, where it became disorganized, forming a teratoma which metastasized to a renal node. The primary tumor was minced and transplanted intraperitoneally; it became an ascites tumor of "embryoid bodies" with yolk sac "rinds" and teratocarcinoma (or embryonal carcinoma) "cores". In 1975, after almost 200 transplant generations in syngeneic hosts, the rinds of some...

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A definitive test for developmental totipotency of mouse malignant teratocarcinoma cells was conducted by cloning singly injected cells in genetically marked blastocysts. Totipotency was conclusively shown in an adult mosaic female whose tumor-strain cells had made substantial contributions to all of the wide range of its somatic tissues analyzed; the clonally propagated cell lineage had therefore differentiated in numerous normal directions. The test cells were from "cores" of embryoid bodies of a euploid, chromosomally male (X/Y), ascites tumor grown only in vivo by transplantation for 8 years. The capacity of cells from the same source to differentiate, in a phenotypic male, into reproductively functional sperms, has been shown in our previous experiments [(1975) Proc. Nat. Acad. Sci. USA 72, 3585-3589]. Cells from this transplant line therefore provide material suitable for projected somatic and germ-line genetic analyses of mammalian differentiation based on "cycling" of mutation-carrying tumor cells through developing embryos. In some animals obtained from single-cell injections tumor-derived cells were sporadically distributed in developmentally unrelated tissues. These cases can be accounted for by delayed and haphazard cellular integration, and by a marked degree of sustained cellular developmental flexibility in early mammalian development, irrespective of certain classical "germ-layer" designations. All mosaic mice obtained have thus far been free of teratomas. In one case, the injected stem cell contributed only to the pancreas and gave rise to a malignancy resembling pancreatic adenocarcinoma. The high modal frequency of euploidy in these individually tested cells thus tends to indicate that a near-normal chromosome complement is sufficient for total restoration of orderly gene expression in a normal embryonic environment; it may also be necessary for teratoma stem-cell proliferation to be terminated there.

Explanted mouse blastocysts were microinjected in the blastocoel cavity with simian virus 40

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Ocular and cutaneous melanomas arose in new inbred lines of transgenic mice having an integrated recombinant gene comprised of the tyrosinase promoter, expressed in pigment cells, and the simian virus 40 early-region transforming sequences. The tumors were hypomelanotic and were histopathologically similar to corresponding human melanomas. Eye melanomas often originated at a young age, chiefly from the retinal pigment epithelium, also from the choroid, and rarely from the ciliary body. The eye tumors grew aggressively, were highly invasive, and metastasized to local and distant sites. The earliest formation of these tumors was associated with higher copy numbers of the transgene; mice of different single-copy lines varied greatly in age of onset and frequency of eye tumors. Coat pigmentation was reduced in almost all lines, to various extents. Primary skin melanomas arose later and less frequently than eye melanomas. Hence they were at early stages and of unknown long-range incidence in this investigation, in which autopsies covered the first half-year of life. For both ocular and cutaneous melanomas, the transgenic mice offer numerous possibilities for experimental study of mechanisms underlying formation and spread of melanomas.Melanomas have a propensity for metastasis (1) We were interested in producing mice with a heritable change, on a uniform genetic background, that would lead consistently to malignant melanomas. We report here the regular occurrence and metastasis of melanomas in inbredstrain transgenic mice with an integrated fusion gene containing the simian virus 40 (SV40) early region under the control of the tyrosinase promoter expressed in pigment cells. This paper deals chiefly with melanomas originating in the eyes and to a lesser extent in the skin, at the age range studied thus far. In the accompanying paper (5), other tumors associated with melanosis occurring in the same animals are described. MATERIALS AND METHODSConstruction and Preparation of Tyr-SV40E. The SV40 early region, including the coding sequences of the transforming large tumor (T) and small tumor (t) antigens (6) and extending from the Avr II (nucleotide 5187) to the BamHI (nucleotide 2533) restriction site, was excised from p6-1AL (a gift from James Alwine, University of Pennsylvania). An Avr II/Bgl II/Sma I adaptor was ligated to the Avr II site, and the fragment was cleaved with Bgl II. Two and one-halfkilobases of 5' flanking sequence of the mouse tyrosinase gene was derived from AgTYR101 (a gift from Siegfried Ruppert; ref. 7) and was used as a promoter. This fragment was bounded by an EcoRI site and a Sau3A site 65 base pairs downstream of the major transcription start site (7, 8) and 15 base pairs upstream of the initiation codon. The tyrosinase promoter was ligated, in the vector pBS, to the Bgl II/BamHI fragment of SV40 early region to generate Tyr-SV40E (Fig. 1). The EcoRI/BamHI fragment containing the mouse tyrosinase promoter and the coding regions of SV40 early genes was separated from vector DNA by gel el...

The zona pellucida may be removed from all stages of the mouse egg by digestion with pronase. Cumulus and corona cells are also dispersed by the enzyme. No change in membrane disgestibility occurs at fertilization. In tests thus far of two-cell eggs and later stages, development continues. Blastocysts exhibit "hatching" behavior despite absence of the zona. Functions of the zona include maintenance of the normal cleavage pattern and prevention of egg fusion.