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1.
Establishment of embryonic stem cell lines from preimplantation mouse embryos homozygous for lethal mutations in the t-complex 总被引:3,自引:0,他引:3
We have determined the frequency at which embryonic stem cell (ESC) lines can be established from inner cell masses (ICMs) isolated from blastocysts homozygous for lethal mutations in the mouse t-complex. Approximately one-third of the expected number, 3/29, of the ESC lines established from embryos obtained by inter-se mating of +/tw18 mice are homozygous for the tw18 haplotype. These tw18/tw18 ESC lines form a variety of cell types in vitro and in vivo, including mesodermal derivatives such as cartilage and muscle. On the basis of these and data from other studies, we suggest that the normal function of the gene represented by the tw18 lethal allele is required for multiplication/survival of mesodermal precursors in the embryo rather than the specification of the mesodermal lineage, and that the lethal effects of this mutation are expressed in only the highly structured environment of the early postimplantation embryo. In studies of the lethal tw5 haplotype, we found that 2/2 ESC lines obtained are mutant homozygotes. Analysis of these data, in conjunction with the results of our earlier study (Magnuson, T., Epstein, C. J., Silver, L. M., and Martin, G. R. (1982), Nature (London) 298, 750-753), suggests that homozygosity for the genes found in the tw5 haplotype does not reduce cell viability. By contrast, 0/16 ESC lines isolated from embryos obtained from matings of +/t0 mice are mutant homozygotes. Analysis of the genotypes of ICM-derived primary stem cell colonies suggests that t0 homozygous ICM cells are unable to undergo sufficient proliferation in vitro to give rise to ESC lines. 相似文献
2.
A method for identifying individual embryos from crosses segregating homozygous t lethals using the marker chromosome Rb7 is described and applied to the tw18 haplotype. In contrast to wild-type and heterozygous littermates, homozygous tw18/tw18 6 and 8 day embryos have very limited growth potentialities in vitro. When transplanted under the testis capsule, homozygous tw18 embryos (3, 6 and 7 day) produce teratomas at a much lower efficiency than heterozygous and wild-type embryos, and the rare teratomas thus obtained have limited growth potentialities when cultured in vitro. Upon transformation with SV40, however, permanent lines of cells of mesodermal origin, capable of myoblastic or adipocytic differentiation, have been obtained. This shows that the effect of the mutation on cell growth can be overcome by SV40 transformation. 相似文献
3.
tclw5 is a t-complex recessive lethal mutation of the tw5-haplotype. Since tw5/tw5 embryos die soon after implantation, the tclw5 gene is thought to play an important role in early embryogenesis. Previous histological studies have demonstrated that tw5 homozygotes do not survive past the gastrulation stage due to extensive death of the embryonic ectoderm, whereas the extraembryonic tissues were less affected. In the present study, we demonstrate that tw5/tw5 embryos may be distinguished from wildtype littermates at embryonic (E) day 5.5. At this stage, the visceral endoderm of tw5/tw5 embryos appeared to be different, possessing smaller and fewer vacuoles compared to normal littermates. This led us to hypothesize that the visceral endoderm may be affected by tclw5. Confirmation was provided by the rescue of tw5/tw5 embryos following aggregation with tetraploid embryos. However, rescued embryos did not survive past E9.0 and displayed an underdeveloped posterior region. This would indicate that the actions of tclw5 extend beyond the midgestation stage. 相似文献
4.
Animalization was induced with evans blue and with Zn++ in embryos of Arbacia punctulata and of Lytechinus variegatus , respectively. Li+ induced vegetalization in A. punctulata embryos. While animalization did not affect the rate of cleavage, vegetalized embryos exhibited a reduction in cell number at post-morula stages. Mid-gastrulae and corresponding experimental embryos each were labeled for 4 hr with uridine-[5-3 H] and with L-[3 H-methyl]-methionine. The rate of uptake of each exogenous RNA precursor was similar in control and in experimental embryos. Purified RNA preparations were fractionated by electrophoresis on 2.4% acrylamide+0.5 % agarose gels. Comparison of rates of incorporation of each RNA precursor into 26s and 18s RNAs indicated that on a per cell basis evans blue- and Zn++ -animalized embryos showed a reduction (0.53–0.56) and Li+ -vegetalized embryos an enhancement (1.41—1.53) in the rate of accumulation of newly made 26s and 18s RNAs compared to controls (1.00). These results suggest that chemically-induced animalized and vegetalized embryos provide useful tools for studying possible differential gene expression in different embryonic germ layers of the developing sea urchin embryo. 相似文献
5.
Binder BJ Landman KA Newgreen DF Simkin JE Takahashi Y Zhang D 《Bulletin of mathematical biology》2012,74(2):474-490
Hindbrain (vagal) neural crest cells become relatively uniformly distributed along the embryonic intestine during the rostral
to caudal colonization wave which forms the enteric nervous system (ENS). When vagal neural crest cells are labeled before
migration in avian embryos by in ovo electroporation, the distribution of labeled neural crest cells in the ENS varies vastly.
In some cases, the labeled neural crest cells appear evenly distributed and interspersed with unlabeled neural crest cells
along the entire intestine. However, in most specimens, labeled cells occur in relatively discrete patches of varying position,
area, and cell number. To determine reasons for these differences, we use a discrete cellular automata (CA) model incorporating
the underlying cellular processes of neural crest cell movement and proliferation on a growing domain, representing the elongation
of the intestine during development. We use multi-species CA agents corresponding to labeled and unlabeled neural crest cells.
The spatial distributions of the CA agents are quantified in terms of an index. This investigation suggests that (i) the percentage
of the initial neural crest cell population that is labeled and (ii) the ratio of cell proliferation to motility are the two
key parameters producing the extreme differences in spatial distributions observed in avian embryos. 相似文献
6.
The tw5 haplotype is a recessive mutation which is lethal when homozygous in mouse embryos following implantation. This series of studies was undertaken to determine the effect of the tw5/tw5 genotype on embryos developing in vitro. Blastocyst embryos from +/tw5 inter se matings were compared with control blastocysts obtained from matings between T/+ and +/+ females and +/tw5 males for their abilities to continue development in vitro in two culture media. The data show that there are no significant differences between the percentages of experimental and control blastocyst embryos which attach and outgrow or which contain inner cell masses on any day of culture up to equivalent gestation day 21 in either media. These findings show that the life span of cells from tw5/tw5 embryos can be extended significantly by in vitro culture. 相似文献
7.
Early C. elegans embryos are transcriptionally active 总被引:3,自引:0,他引:3
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10.
Il-Kuk Chang Atsushi Yoshiki Moriaki Kusakabe Atsushi Tajima Tateki Chikamune Mitsuru Naito Tadao Ohno 《Cell biology international》1995,19(7):569-576
Intrinsic primordial germ cells (PGCs) from stage 27 (5-day-old) chick embryonic germinal ridges were cultured in vitro for a further 5 days, and shown to proliferate on stroma cells derived from the germinal ridge. To determine whether these cultured PGCs could colonize and contribute to the germ-line, PGCs were isolated by gentle pipetting, labeled with PKH26 fluorescent dye and injected into the blood stream of stage 17 (2.5-day-old) chick embryos. The recipient embryos were incubated until they reached stage 28. Thin sections of these embryos were analysed by fluorescent confocal laser microscopy. These analyses showed that the labeled donor PGCs had migrated into the germinal ridges of the recipient embryos, and transplanted PGCs had undergone at least 3-7 divisions. These results suggest that PGCs that had passed far beyond the migration stage in vivo were still able to migrate, colonize and proliferate in recipient chick embryonic gonads. 相似文献
11.
Abstract: To investigate certain biochemical aspects of myelination, a study was undertaken of the messenger-like RNA in the nervous system of pre- myelinating 14-day embryos and of myelinating 17-day embryos and 3-day chicks. The central and peripheral nervous systems of the chick were found to contain and to actively synthesize poly(A)+ RNA. RNA species binding to oligo(dT)-cellulose contained a relatively high proportion of adenylate residues and were resistant to the actions of pancreatic and T1 ribonucleases. Preparations labeled by incubation with adenosine in vitro showed a decrease in the proportion of poly(A)+ RNA as the age of the animal increased, while preparations labeled in vivo exhibited the opposite trend. Polyacrylamide gel electrophoretograms of both in vivo and in vitro labeled pqeparations showed that the poly(A)+ fractions contained mainly heterodisperse RNA species. The average molecular size of poly(A)+ RNAs of purified polysomal fractions of nerve RNA from 3-day chicks was smaller than 18S, whereas that of total poly(A)− RNA was larger than 18s. The proportion of poly(A)+ molecules larger than 18s was lower in the rapidly myelinating nerve tissues of 17-day embryos and post-hatching chicks than in those of premyelinating 14-day embryos. Similar results were obtained for crude nuclear RNA fractions or RNA preparations fractionated under denaturing conditions. These results are consistent with previous work showing that the embryonic peripheral nerve contains a larger proportion of high-molecular-weight, messenger-like RNA molecules than does nerve tissue from young chicks or adults. 相似文献
12.
New alleles of brachyury (Tkt1, Tkt4) were induced in the mouse complete tw5 haplotype by ethylnitrosourea (ENU). Like the original brachyury (T) mutation, the new alleles cause a short-tailed phenotype in heterozygotes, and interact with the t complex tail interaction factor (tct) in trans to cause phenotypically tailless mice. Because ENU is mainly a point mutagen, it is important to determine that the new alleles are homozygous embryonic lethal mutations like the original T allele, and to characterize their embryonic lethal phenotype. Moreover, the Tkt1 mutation maps to an inverted position relative to quaking (qk) in t haplotypes as compared with its position on normal chromosome 17. The Tkt1 allele was separated from the resident tw5 lethal gene, tclw5, by recombination, allowing embryology studies to be performed. Embryological analyses show that the Tkt1 allele is nearly identical to the classic T allele. At 9 and 10 days of development, homozygous Tkt1/Tkt1 embryos are grossly abnormal with properties including 1) irregular, disorganized somite pairs, 2) a shortened posterior end of the embryo, 3) an irregular neural tube, and 4) an abnormal notochord. In addition, 10 day-old abnormal embryos have anterior limb buds that point dorsally rather than ventrally, and are smaller than normal littermates. We conclude that the Tkt1 mutation is a valuable allele for both mapping and molecular characterization of the brachyury locus. 相似文献
13.
Cell fate in the polar trophectoderm of mouse blastocysts as studied by microinjection of cell lineage tracers 总被引:2,自引:0,他引:2
Horseradish peroxidase (HRP), together with Fast Green or rhodamine-conjugated dextran (RDX), was used as an intracellular lineage tracer to determine cell fate in the polar trophectoderm of 3.5-day-old mouse embryos. In HRP-injected midstage (approximately 39-cell) and expanded (approximately 65-cell) blastocysts incubated for 24 hr, the central polar trophectoderm cell was displaced from the embryonic pole an average of 20 micron (5% of blastocyst circumference) and 29 micron (6% of blastocyst circumference), respectively. Expanded blastocysts injected with HRP + Fast Green and incubated for 24 hr or with HRP + RDX and incubated for 48 hr showed a displacement of 24 micron (4% of blastocyst circumference) and 88 micron (14% of blastocyst circumference), respectively. Up to 10 HRP-positive trophectoderm cells were observed among embryos incubated for 48 hr, indicating that in those cases, the labeled progenitor cells had divided at least three times. Our observations show that the central polar trophectoderm cell divides in the plane of the trophectoderm in expanded blastocysts and, along with its descendants, is displaced toward the mural trophectoderm. The systematic tandem displacement of labeled cells and their descendants toward the abembryonic pole suggests the presence of a proliferative area at the embryonic pole of the blastocyst. Large shifts in inner cell mass (ICM) position in relation to the trophectoderm do not occur during blastocyst expansion. Furthermore, random movements within the polar trophectoderm population do not account for the replacement of labeled cells by unlabeled polar trophectoderm cells. Rather, we propose the hypothesis that the ICM contributes these replacement cells to the polar trophectoderm during blastocyst expansion. 相似文献
14.
On the isolation of embryonic stem cells: Comparative behavior of murine, porcine and ovine embryos 总被引:9,自引:0,他引:9
The efficiency of isolation and the characteristics of embryo-derived cell lines from murine, porcine, and ovine embryos cultured on STO feeders or homologous embryonic fibroblasts (HEF) feeders were compared. While murine isolated ICM or intact embryos plated on STO or HEF feeders gave rise to cell lines with embryonic stem cell-like (ES-like) morphology, ovine embryos did not. Cell lines with ES-like morphology were isolated from porcine intact embryos and isolated ICM when plated on STO feeders but not when plated on HEF. Neither murine nor porcine ES-like cell lines expressed cytokeratin 18 or vimentin. Unlike murine ES-like cell lines, porcine ES-like cells did not undergo observable differentiation in vitro or in vivo. Cell lines with epithelial-like morphology were isolated from porcine and ovine embryos. Both porcine and ovine epithelial-like cell kines expressed cytokeratin 18. When induced to differentiate in vitro, porcine and ovine epithelial-like cell lines formed vesicular structures. Electron microscopy revealed that the porcine vesicles were composed of polarized epithelial cells, each with a basally-located nucleus and an apical border containing numerous microvilli with a well organized microfilament core. The results of this study show that conditions which allow isolation of ES cells from murine embryos allow the isolation of porcine embryo-derived cell lines sharing some, but not all, the characteristics of murine ES cells. 相似文献
15.
《The Journal of cell biology》1984,99(4):1343-1353
Certain sperm components labeled with fluorescein isothiocyanate or its radioactive derivative, 125I-diiodofluorescein isothiocyanate (125IFC), are transferred at fertilization to the egg, where they persist throughout early cleavage stages at a localized site in the embryo cytoplasm (Gabel, C. A., E. M. Eddy, and B. M. Shapiro, 1979, Cell, 18:207-215; Gundersen, G. G., C. A. Gabel, and B. M. Shapiro, 1982, Dev. Biol., 93:59-72). By using image intensification we have extended these observations in the sea urchin to the pluteus larval stage, in which greater than 60% of the embryos have localized fluorescent sperm components. Because of the unusual persistence of the sperm components in the embryo, a characterization of the nature of the labeled species in sea urchin sperm was undertaken. Approximately 10% of the 125IFC was in sperm polypeptides of Mr greater than 15,000. These proteins were on the sperm surface as shown by their sensitivity to externally added proteases. The remainder of the 125IFC in sperm was in several low- molecular-weight species, none of which was 125IFC-derivatized phospholipid. To determine if any labeled sperm polypeptides remained intact in the embryo after fertilization, 125IFC-labeled sperm proteins were recovered from one-cell and late gastrula stage embryos by using an anti-IFC immunoadsorbent. Most of the labeled sperm proteins were degraded shortly after fertilization; however, distinct sets of labeled polypeptides were recovered from both one-cell and gastrula stage embryos. Six of the labeled polypeptides recovered from both embryonic stages had identical SDS gel mobilities as labeled sperm polypeptides. Other polypeptides in the embryos appeared to arise from limited proteolysis of sperm proteins. Thus, in this physiological cell fusion system, individual sperm proteins are transferred to the egg at fertilization, and some persist intact or after specific, limited degradation long after gamete fusion, until at least the late gastrula stage. 相似文献
16.
We have previously shown that the targeted deletions of both type I keratins (K) 18 and 19 cause lethality by embryonic day (e) 9.5 due to fragility and cytolysis of trophoblast giant cells. The development of the embryo proper appeared to be unaffected and its death was caused by nutrient deficiency. In order to address the function of keratins within the embryo proper, lethality due to extraembryonic tissue failure must be overcome. One approach to rescue doubly deficient embryos is by aggregating knockout embryos with tetraploid wild-type embryos. As a general tool, tetraploid aggregation can be used to rescue embryonic lethality caused by defects in extraembryonic tissues like the placenta, trophoblast or yolk sac. We rescued K18-/- K19-/- embryos until e11.5, using this approach, proving that the loss of the keratin cytoskeleton causes defects in the trophoblast giant cell layer, but has no effect on early development of the embryo proper. 相似文献
17.
The murine t complex on chromosome 17 contains a number of homozygous lethal and semi-lethal mutations that disrupt development of the mouse embryo. We recently characterized an embryonic lethality in the rat that results from a germ-line mutation in the tuberous sclerosis 2 (Tsc-2) tumour suppressor gene (the Eker mutation). Remarkably, mouse embryos homozygous for tw8 mutation display cranial defects reminiscent of those observed in rat embryos homozygous for the Eker mutation. To determine whether the Tsc-2 gene, which is in the t complex, is mutated in tw8 or other t haplotypes, we characterized this gene in a series of t haplotype mice. Four Tsc-2 polymorphisms were identified: three in the coding region and one intronic that appeared to be common to all t haplotypes analysed. No evidence was found to argue that the Tsc-2 gene is altered in tw8 haplotype mice. However, in the tw5 haplotype we found a G to T mutation in Tsc-2 that was present only in this t haplotype. In contrast to other polymorphisms within the Tsc-2 coding region which did not result in amino acid changes in Tsc-2 gene product tuberin, this mutation substituted a phenylalanine for a conserved cysteine in tw5 tuberin. Within the t complex, the Tsc-2 gene and the putative tw5 locus appeared to map to different positions, complicating identification of Tsc-2 as a candidate for the tw5 locus and suggesting that the G to T mutation in the Tsc-2 gene may have arisen independently of the tw5 functional mutation. 相似文献
18.
R. S. Canseco A. E. T. Sparks R. L. Page C. G. Russell J. L. Johnson W. H. Velander R. E. Pearson W. N. Drohan F. C. Gwazdauskas 《Transgenic research》1994,3(1):20-25
Litter size of DNA microinjected zygotes is lower than for non-manipulated zygotes. The rate of embryonic and fetal survival in early, mid and late gestation was determined to assess whether DNA integration was responsible for embryonic losses. Also, the effect of including non-microinjected embryos with injected embryos on pregnancy rate and transgenic pup production was determined. In Experiment 1, one-cell embryos from immature CD-1 mice were microinjected with a whey acidic protein promoter-human protein C gene construct. One hour after microinjection embryos were transferred to pseudopregnant recipients (45 transfers of 30 embryos each). Fifteen recipients were sacrificed on day 4, 12 and 18 of gestation and the embryos/fetuses analysed for the transgene. The percentage of embryos or fetuses that were positive for the transgene was not significantly different at any day. However, the number of viable embryos at day 4 was significantly greater than fetuses on days 12 or 18. In addition, a high degree of mosaicism was observed in day 18 fetuses and placentae recovered. In Experiment 2, one-cell embryos from CD-1 mice were microinjected and co-transferred with non-manipulated embryos (C57BL/6). Pregnancy rate and the total number of pups born were improved by addition of non-injected embryos. However, the number of transgenic mice produced was similar whether non-injected embryos were included or not. There were 32.2% (15/46) transgenic pups when 0 non-injected embryos were transferred compared with 15.1% (13/86) transgenic pups when 4 or 8 non-injected embryos were added to the transfers. In summary, a high degree of embryonic and fetal mortality occurs among microinjected embryos. Furthermore, since the percentage of transgenesis did not change throughout pregnancy, DNA integration does not appear to account for all of the embryonic losses. other factor(s) related to the microinjection procedure may be involved in the embryonic and fetal failure of microinjected embryos. Addition of non-injected embryos, although it increased pregnancy rate and the number of pups born from microinjected embryos, actually decreased the number of transgenic pups obtained per pregnancy. 相似文献
19.
Day 10 rat embryos were exposed in vitro to [chloroethyl 3H] cyclophosphamide (3H-CP) at 4 micrograms/ml over a 24-hour period and the uptake and binding of labeled drug were monitored autoradiographically and biochemically. Autoradiographic analysis revealed that embryos exposed to 3H-CP and a complete activating system exhibited radioactivity distributed throughout the embryo. Subsequent analysis indicated that the distribution of autoradiographic grains on a per cell basis ranged from 7.7 in surface ectoderm to 13.4 in the neuroepithelium. No correlation was found between the sensitivity of various embryonic tissues to the cytotoxic effects of CP and the number of grains per cell. Direct radiochemical analysis of the amount of tritium taken up and bound by embryos under bioactivating conditions (3H-CP + S-9 + cofactors) confirmed the autoradiographic analysis. Autoradiographic and radiochemical analyses demonstrate that embryos exposed under bioactivating conditions take up and bind approximately three times more tritium than embryos exposed under nonactivating conditions (3H-CP + S-9 without cofactors). Additional studies have demonstrated that uptake and binding of tritium from bioactivated 3H-CP only are linear over the first 10 hours of incubation with no detectable increases thereafter. 相似文献
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