scholarly journals Mcl-1 deficiency results in peri-implantation embryonic lethality

2000 ◽  
Vol 14 (1) ◽  
pp. 23-27 ◽  
Author(s):  
Julie L. Rinkenberger ◽  
Susan Horning ◽  
Barbara Klocke ◽  
Kevin Roth ◽  
Stanley J. Korsmeyer
Keyword(s):  
Family Member ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
In Utero ◽  
Embryonic Lethality ◽  
Preimplantation Development

We disrupted the Mcl-1 locus in murine ES cells to determine the developmental roles of this Bcl-2 family member. Deletion of Mcl-1 resulted in peri-implantation embryonic lethality. Mcl-1−/− embryos do not implant in utero, but could be recovered at E3.5–4.0. Null blastocysts failed to hatch or attach in vitro, indicating a trophectoderm defect, although the inner cell mass could grow in culture. Of note, Mcl-1−/−blastocysts showed no evidence of increased apoptosis, but exhibited a delay in maturation beyond the precompaction stage. This model indicates that Mcl-1 is essential for preimplantation development and implantation, and suggests that it has a function beyond regulating apoptosis.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

CSF-1 and mouse preimplantation development in vitro

Development ◽  
1995 ◽  
Vol 121 (5) ◽  
pp. 1333-1339 ◽  
Author(s):  
P. Bhatnagar ◽  
V.E. Papaioannou ◽  
J.D. Biggers
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Preimplantation Development ◽  
High Rate ◽  
Colony Stimulating Factor ◽  
Trophoblast Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The effects of macrophage colony stimulating factor on the development of the zygote to the blastocyst stage of an outbred strain of mouse have been studied in KSOM, an improved medium that supports a high rate of in vitro development. Macrophage colony stimulating factor accelerates the formation of the blastocyst cavity by day 4 (96 hours post-hCG). It also increases overall embryonic cell number through a differential increase in the number of trophoblast cells, with no significant effect on the number of inner cell mass cells. By day 5 of culture (120 hours post-hCG), colony stimulating factor-treated embryos have about 20 more trophoblast cells than control embryos, an increase of about 30 percent of the total number of cells in a control blastocyst. The maximum response of embryos was obtained at a concentration around 540 U ml-1 colony stimulating factor (identical to 918 Stanley units ml-1), and the cytokine can produce the same effects even if it is present in the medium for only part of the culture period. This in vitro stimulation of preimplantation development with macrophage colony stimulating factor is compatible with continued normal fetal development in vivo.

Investigation of the determinative state of the mouse inner cell mass

Development ◽  
1975 ◽  
Vol 33 (4) ◽  
pp. 979-990
Author(s):  
J. Rossant
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
In Utero ◽  
Glucose Phosphate Isomerase ◽  
Trophoblast Cells ◽  
Electrophoretic Variants ◽  
Glucose Phosphate

Inner cell masses (ICMs) were dissected from 3½- and 4½-day blastocysts and cultured in contact with 2½-day morulae. Blastocysts and morulae were homozygous for different electrophoretic variants of the enzyme glucose phosphate isomerase (GPI). Aggregation of ICMs and morulae was observed, and such aggregates were able to form blastocysts in vitro and morphologically normal foetuses in utero. GPI analysis of these conceptuses revealed that most were chimaeric. However, donor ICM-type isozyme was only detected in the embryonic and extra-embryonic fractions of the chimaeras and never in the trophoblastic fraction. Thus, ICM cells appear unable to form trophoblast derivatives even when exposed to ‘outside’ conditions as experienced by developing trophoblast cells. This is evidence that ICM cells, although not overtly differentiated, are determined by 3½ days.

183 DEVELOPMENT OF PORCINE EMBRYOS MICROINJECTED WITH PORCINE EMBRYONIC GERM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 199
Author(s):  
C.-H. Park ◽  
S.-G. Lee ◽  
D.-H. Choi ◽  
M.-G. Kim ◽  
C. K. Lee
Keyword(s):  
Germ Cells ◽  
Fluorescent Protein ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Green Fluorescent Protein Gene ◽  
Cleavage Stage ◽  
Allocation Pattern

Embryonic germ (EG) cells, derived from primordial germ cells in the developing fetus, are similar to embryonic stem (ES) cells in terms of expression pattern of undifferentiated markers and their ability to colonize both the somatic and the germ cell lines following injection into a host blastocyst, which has been proven in mouse. Several studies using porcine EG cells have shown that it is possible to produce somatic chimeras after blastocyst injection. However, not only was the degree of reported chimerism low, but also there has been no report about the fate of injected EG cells in porcine blastocysts. This study was designed to observe the distribution pattern of porcine EG cells in chimeric blastocyst after injection into cleavage-stage porcine embryos. To ascertain development of microinjected porcine embryos with EG cells, 10 to 15 EG cells were injected into cleavage stage of in vitro fertilized embryos and cultured up to blastocyst. Also, porcine EG cells were labeled with DiO (Invitrogen, Carlsbad, CA) on the cell membrane or transfected with green fluorescent protein gene to observe whether the EG cells injected in the host embryo would incorporate into the inner cell mass (ICM) or trophectoderm (TE). Chimeric embryos were produced and allowed to develop into blastocysts to investigate the injected EG cells would come to lie in ICM and/or TE of the blastocyst, by scoring their position. In result, developmental rate was similar in all treatments. In all treatments, EG cells were mainly allocated in both ICM and TE of the chimeric blastocysts. These results suggest that examining the allocation pattern of injected EG cells, maintained pluripotency in vitro, could provide clues of differentiation process in vivo. Furthermore, to enhance the allocation of EG cells into the embryonic lineage, it would be required to optimize the culture condition for EG cells as well as embryos. Further experiment are needed to determine whether the injected EG cells could maintain their properties throughout the environment in the embryonic development in vitro. Table 1. Distribution of the porcine EG cells microinjected into cleavage-stage embryos

scholarly journals Localization of nitric oxide synthase activity in unfertilized oocytes and fertilized embryos during preimplantation development in mice

Reproduction ◽  
2001 ◽  
pp. 957-963 ◽  
Author(s):  
A Nishikimi ◽  
T Matsukawa ◽  
K Hoshino ◽  
S Ikeda ◽  
Y Kira ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Preimplantation Development ◽  
Cell Stage ◽  
Nos Inhibitors ◽  
Development In Vitro ◽  
Oxide Synthase

Changes in the activities of nitric oxide synthase (NOS) during embryonic development, and the distribution of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) isoforms were examined in unfertilized mouse oocytes at the second meiotic metaphase (MII) stage and in fertilized mouse embryos during preimplantation development. In addition, the effects of NOS inhibitors on mouse preimplantation development in vitro were investigated. The activities of NOS in MII oocytes and fertilized embryos during the preimplantation period were determined by NADPH-diaphorase staining. Although NOS activity was detected in unfertilized MII oocytes, the intensity of staining was much weaker than that of fertilized embryos at the one-cell stage. There was a decrease in NOS activity in embryos from the four-cell to the eight-cell stage; however, NOS activity increased again in embryos at the morula stage, particularly in the inner cell population. In the expanded blastocysts, staining was confined to the inner cell mass. Immuno-cytochemical staining showed that eNOS and iNOS were expressed in the cytoplasm of oocytes and embryos during the preimplantation period, and eNOS was also distributed in the nuclei of the embryos. When one-cell embryos were treated with 1 mmol N(omega)-nitro-L-arginine methyl ester (L-NAME) l(-1), their development in vitro was arrested at the two-cell stage. This inhibition of development was overcome by the addition of 1 mmol L-arginine l(-1) to the medium. These observations indicate that nitric oxide plays an important role as a diffusible regulator of cell proliferation and differentiation, especially at the developmental transition from the two-cell to the four-cell stage during preimplantation development of mice.

scholarly journals OCT4/POU5F1 is required for NANOG expression in bovine blastocysts

2018 ◽  
Vol 115 (11) ◽  
pp. 2770-2775 ◽  
Author(s):  
Kilian Simmet ◽  
Valeri Zakhartchenko ◽  
Julia Philippou-Massier ◽  
Helmut Blum ◽  
Nikolai Klymiuk ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Preimplantation Development ◽  
Null Mouse ◽  
General Strategy ◽  
Vitro Fertilization ◽  
Domestic Species ◽  
Oct4 Protein ◽  
Salt And Pepper

Mammalian preimplantation development involves two lineage specifications: first, the CDX2-expressing trophectoderm (TE) and a pluripotent inner cell mass (ICM) are separated during blastocyst formation. Second, the pluripotent epiblast (EPI; expressing NANOG) and the differentiated primitive endoderm (PrE; expressing GATA6) diverge within the ICM. Studies in mice revealed that OCT4/POU5F1 is at the center of a pluripotency regulatory network. To study the role of OCT4 in bovine preimplantation development, we generated OCT4 knockout (KO) fibroblasts by CRISPR-Cas9 and produced embryos by somatic cell nuclear transfer (SCNT). SCNT embryos from nontransfected fibroblasts and embryos produced by in vitro fertilization served as controls. In OCT4 KO morulae (day 5), ∼70% of the nuclei were OCT4 positive, indicating that maternal OCT4 mRNA partially maintains OCT4 protein expression during early development. In contrast, OCT4 KO blastocysts (day 7) lacked OCT4 protein entirely. CDX2 was detected only in TE cells; OCT4 is thus not required to suppress CDX2 in the ICM. Control blastocysts showed a typical salt-and-pepper distribution of NANOG- and GATA6-positive cells in the ICM. In contrast, NANOG was absent or very faint in the ICM of OCT4 KO blastocysts, and no cells expressing exclusively NANOG were observed. This mimics findings in OCT4-deficient human blastocysts but is in sharp contrast to Oct4-null mouse blastocysts, where NANOG persists and PrE development fails. Our study supports bovine embryogenesis as a model for early human development and exemplifies a general strategy for studying the roles of specific genes in embryos of domestic species.

W41/W41 blastocyst complementation: a system for genetic modeling of hematopoiesis

Blood ◽  
2010 ◽  
Vol 115 (1) ◽  
pp. 47-50 ◽  
Author(s):  
Lina Jansson ◽  
Jonas Larsson
Keyword(s):  
Inner Cell Mass ◽  
Fetal Liver ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Mouse Strains ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Blastocyst Complementation

Abstract We report a rapid and highly efficient approach to generate mice in which the hematopoietic system is derived from embryonic stem (ES) cells. We show that ES cells injected into blastocysts from the c-kit–deficient W41/W41 mouse strain have a clear advantage over the W41/W41 blastocyst-derived inner cell mass cells in founding the hematopoietic system. Fetal liver hematopoietic stem cells from W41/W41 blastocyst complementation embryos can be transplanted to establish large cohorts of bone marrow chimeras with hematopoiesis of practically pure ES-cell origin. Using ES cells with site-directed modifications, we show how this system can be used to drive inducible transgene expression in hematopoietic cells in a robust and reliable manner both in vitro and in vivo. The approach avoids the cost and time constraints associated with the creation of standard transgenic mouse strains while taking advantage of the sophisticated site-directed manipulations that are possible in ES cells.

scholarly journals Hydroxysteroid (17β) Dehydrogenase 12 Is Essential for Mouse Organogenesis and Embryonic Survival

Endocrinology ◽  
2010 ◽  
Vol 151 (4) ◽  
pp. 1893-1901 ◽  
Author(s):  
Pia Rantakari ◽  
Heidi Lagerbohm ◽  
Mika Kaimainen ◽  
Jukka-Pekka Suomela ◽  
Leena Strauss ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Neuronal Development ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Lacz Gene ◽  
Developmental Defects ◽  
Proliferation Capacity

Hydroxysteroid (17β) dehydrogenases (HSD17Bs) have a significant role in steroid metabolism by catalyzing the conversion between 17-keto and 17β-hydroxysteroids. However, several studies in vitro have shown that some of these enzymes may also be involved in other metabolic pathways. Among these enzymes, HSD17B12 has been shown to be involved in both the biosynthesis of estradiol and the elongation of the essential very long fatty acids in vitro and in vivo. To investigate the function of mammalian HSD17B12 in vivo, we generated mice with a null mutation of the Hsd17b12 gene (HSD17B12KO mice) by using a gene-trap vector, resulting in the expression of the lacZ gene of the trapped allele. The β-galactosidase staining of the heterozygous HSD17B12KO mice revealed that Hsd17b12 is expressed widely in the embryonic day (E) 7.5-E9.5 embryos, with the highest expression in the neural tissue. The HSD17B12KO mice die at E9.5 at latest and present severe developmental defects. Analysis of the knockout embryos revealed that the embryos initiate gastrulation, but organogenesis is severely disrupted. As a result, the E8.5-E9.5 embryos were void of all normal morphological structures. In addition, the inner cell mass of knockout blastocysts showed decreased proliferation capacity in vitro, and the amount of arachidonic acid was significantly decreased in heterozygous HSD17B12 ES cells. This, together with the expression pattern, suggests that in mouse, the HSD17B12 is involved in the synthesis of arachidonic acid and is essential for normal neuronal development during embryogenesis.

275 DERIVATION OF PLURIPOTENT CELL LINES FROM PIG EMBRYOS: IN VITRO-FERTILIZED V. PARTHENOGENTIC ACTIVATION

2009 ◽  
Vol 21 (1) ◽  
pp. 235
Author(s):  
F. Gandolfi ◽  
G. Pennarossa ◽  
L. Attanasio ◽  
S. Antonini ◽  
B. Gasparrini ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Telomerase Activity ◽  
Animal Biotechnology ◽  
Pluripotency Markers ◽  
Biology Research ◽  
Parthenogenetic Embryos

The establishment of porcine pluripotent ES cell lines would be an exciting and novel tool for animal biotechnology, such as cloning and transgenesis. Furthermore, it would represent a useful model for biomedical research, cell therapy, xenotransplantation as well as developmental biology research. However, in spite of several studies, no conclusive results have been obtained and a number of technical questions are still to be answered in order to derive genuine ES cells in the pig. Here we report the results obtained in our laboratory aimed at comparing IVF v. parthenogenetic embryos as a source for the establishment of putative ES cells. Oocytes were divided in two groups and subjected to IVF or parthenogenetically activated with ionomicyn and 6-DMAP. They were cultured in NCSU for 7 days and then subjected to immuno-surgery. Inner cell mass were plated onto inactivated STO feeder cells and outgrowth formation was monitored. Cells were passaged to a new STO monolayer every 7 days. Assessment of pluripotency markers was carried out both by RT-PCR and immunocytochemical analysis at every passage for up to 22 passages. Telomerase activity was measured every 5 passages. The results indicate that parthenogenetic embryos, although less resilient than IVF embryos to immuno-surgery, have a significantly greater ability to generate outgrowths and stable cell lines. Moreover, 77% of the 39 parthenogenetic lines derived v. only 33% of the IVF ones expressed pluripotency markers and displayed high telomerase activity. Altogether our findings are consistent with data obtained in the human where the efficiency to derive hES cell lines from parthenogenetic blastocysts appears greater as compared with regular blastocysts from IVF embryos (Cheng L 2008 Cell Research 18, 215–217). Table 1. Supported by: Prin 2005, 2006.

scholarly journals Generation of embryonic stem cell lines from mouse blastocysts developed in vivo and in vitro: relation to Oct-4 expression

Reproduction ◽  
2006 ◽  
Vol 132 (1) ◽  
pp. 59-66 ◽  
Author(s):  
S Tielens ◽  
B Verhasselt ◽  
J Liu ◽  
M Dhont ◽  
J Van Der Elst ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Stem Cell Lines

Embryonic stem (ES) cells are the source of all embryonic germ layer tissues. Oct-4 is essential for their pluripotency. Sincein vitroculture may influence Oct-4 expression, we investigated to what extent blastocysts culturedin vitrofrom the zygote stage are capable of expressing Oct-4 and generating ES cell lines. We comparedin vivowithin vitroderived blastocysts from B6D2 mice with regard to Oct-4 expression in inner cell mass (ICM) outgrowths and blastocysts. ES cells were characterized by immunostaining for alkaline phosphatase (ALP), stage-specific embryonic antigen-1 (SSEA-1) and Oct-4. Embryoid bodies were made to evaluate the ES cells’ differentiation potential. ICM outgrowths were immunostained for Oct-4 after 6 days in culture. A quantitative real-time PCR assay was performed on individual blastocysts. Of thein vitroderived blastocysts, 17% gave rise to ES cells vs 38% of thein vivoblastocysts. Six-day old outgrowths fromin vivodeveloped blastocysts expressed Oct-4 in 55% of the cases vs 31% of thein vitroderived blastocysts. The amount of Oct-4 mRNA was significantly higher for freshly collectedin vivoblastocysts compared toin vitrocultured blastocysts.In vitrocultured mouse blastocysts retain the capacity to express Oct-4 and to generate ES cells, be it to a lower level thanin vivoblastocysts.

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