Generation of mice derived from embryonic stem cells using blastocysts of different developmental ages

Ohta, Hiroshi; Sakaide, Yuko; Wakayama, Teruhiko

doi:10.1530/rep-08-0184

Cited by 13 publications

(14 citation statements)

References 17 publications

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“…6D), or placental hypertrophy phenotypes. Our average ntES-4N pup body and placenta weights from the LDLr KO ntES cell line are much lower than weights reported for ntES-4N pups by Ohta et al [16,18], who observed average body and placenta weights of approximately 1.6 and 0.2 g, respectively, for their ntES-4N pups. The difference may be attributed to the different mouse strains used, inbred C57BL/6 ntES cells in our study, and the mostly hybrid strains, B6129 F1, in theirs.…”

Section: Discussioncontrasting

confidence: 79%

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Acceptance of Embryonic Stem Cells by a Wide Developmental Range of Mouse Tetraploid Embryos1

Lin

Amano

Zhang

et al. 2010

Biology of Reproduction

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Tetraploid (4N) complementation assay is regard as the most stringent characterization test for the pluripotency of embryonic stem (ES) cells. The technology can generate mice fully derived from the injected ES cell (ES-4N) with 4N placentas. However, it remains a very inefficient procedure owing to a lack of information on the optimal conditions for ES incorporation into the 4N embryos. In the present study, we injected ES cells from embryos of natural fertilization (fES) and somatic cell nuclear transfer (ntES) into 4N embryos at various stages of development to determine the optimal stage of ES cells integration by comparing the efficiency of full-term ES-4N mouse generation. Our results demonstrate that fES/ntES cells can be incorporated into 4N embryos at 2-cell, 4-cell and blastocyst stages and full-term mice can be generated. Interestingly, ntES cells injected into the 4-cell group resulted in the lowest efficiency (5.6%) compared to the 2-cell (13.8%, P > 0.05) and blastocyst (16.7%, P < 0.05) stages. Because 4N embryos start to form compacted morulae at the 4-cell stage, we investigated whether the lower efficiency at this stage was due to early compaction by injecting ntES cells into artificially de-compacted embryos treated with calcium free medium. Although the treatment changed the embryonic morphology, it did not increase the efficiency of ES-4N mice generation. Immunochemistry of the cytoskeleton displayed microtubule and microfilament polarization at the late 4-cell stage in 4N embryos, which suggests that de-compaction treatment cannot reverse the polarization process. Taken together, we show here that a wide developmental range of 4N embryos can be used for 4N complementation and embryo polarization and compaction may restrict incorporation of ES cells into 4N embryos.

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Section: Discussioncontrasting

confidence: 79%

“…Because of the low efficiency of 4N complementation, attempts to improve it had been made by using later-stage embryos at E3.5-5.5 of pregnancy, equivalent to expanded to hatched blastocysts [16]. It was shown that injection of ES cells into E4.5 embryos gave the highest efficiency.…”

Section: Discussionmentioning

confidence: 99%

Acceptance of Embryonic Stem Cells by a Wide Developmental Range of Mouse Tetraploid Embryos1

Lin

Amano

Zhang

et al. 2010

Biology of Reproduction

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“…Because this is an un-expected outcome and it is out of the scope of this study to identify the exact cause for the abnormal morphology of iPS-4N pups. It may be related to the intrinsic characteristics of ES-4N mice, as has been suggested by previous reports [34], [35], although we didn’t observe the hernia phenotype in the rest of our pluripotent cell lines. Another possible explanation is mutagenesis by retrovirus insertions during the iPS induction process.…”

Section: Discussionsupporting

confidence: 54%

Improved Derivation Efficiency and Pluripotency of Stem Cells from the Refractory Inbred C57BL/6 Mouse Strain by Small Molecules

2014

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The ability of small molecules to maintain self-renewal and to inhibit differentiation of pluripotent stem cells has been well-demonstrated. Two widely used molecules are PD 98059 (PD), an inhibitor of extracellular-signal-regulated kinase 1 (ERK), and SC1 (Pluripotin), which inhibits the RasGAP and ERK pathways. However, no studies have been conducted to compare their effects on the pluripotency and derivation of embryonic stem (ES) cells from inbred mice C57BL/6, an important mouse strain frequently used to model behavior, cognitive functions, immune system, and metabolic disorders in humans and also the first mouse strain chosen to be sequenced for its entire genome. We found significantly increased derivation efficiency of ES cells from in vivo fertilized embryos (fES) of C57BL/6 with the use of PD (71.4% over the control of 35.3%). Because fES and ES from cloned embryos (ntES) are not distinguishable in transcription or translation profiles, we used ntES cells to compare the effect of small molecules on their in vitro characteristics, in vitro differentiation ability, and the ability to generate full-term ntES-4N pups by tetraploid complementation. NtES cells exhibited typical ES characteristics and up-regulated Sox2 expression in media with either small-molecule. Higher rates of full term ntES-4N pup were generated by the supplementation of PD or SC1. We obtained the highest efficiency of ntES-4N pup generation ever reported from this strain by supplementing ES medium with SC1. Lastly, we compared the pluripotency of fES, ntES and induced pluripotent stem (iPS) cells of C57BL/6 background using the tetraploid complementation assay. A significant increase in implantation sites and the number of full-term pups were obtained when fES, ntES, and iPS cells were cultured with SC1 compared to the control ES medium. In conclusion, supplementing ES cell culture medium with PD and SC1 increases the derivation efficiency and pluripotency, respectively, of stem cells derived from the refractory inbred C57BL/6 strain.

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“…The combined use of targeted insertion of a transgene or shRNA into the ROSA26 locus using RMCE or into the COL1A1 locus using FRT-mediated targeting and subsequent ES cell injection using tetraploid blastocysts significantly accelerates the generation of transgenic or knockdown mice (Seibler et al ., 2003; Mackay and West, 2005; Ohta et al ., 2008). Only 2 weeks are needed for cloning the DNA vector, 3 weeks are needed for generating targeted ES cell clones, and 4 weeks are needed for producing newborn mice.…”

Section: Combined Use Of Targeted Transgenesis and Tetraploid Blastocmentioning

confidence: 99%

Genetically Engineered Mouse Models for Drug Development and Preclinical Trials

Lee

2014

Biomolecules & Therapeutics

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Drug development and preclinical trials are challenging processes and more than 80% to 90% of drug candidates fail to gain approval from the United States Food and Drug Administration. Predictive and efficient tools are required to discover high quality targets and increase the probability of success in the process of new drug development. One such solution to the challenges faced in the development of new drugs and combination therapies is the use of low-cost and experimentally manageable in vivo animal models. Since the 1980’s, scientists have been able to genetically modify the mouse genome by removing or replacing a specific gene, which has improved the identification and validation of target genes of interest. Now genetically engineered mouse models (GEMMs) are widely used and have proved to be a powerful tool in drug discovery processes. This review particularly covers recent fascinating technologies for drug discovery and preclinical trials, targeted transgenesis and RNAi mouse, including application and combination of inducible system. Improvements in technologies and the development of new GEMMs are expected to guide future applications of these models to drug discovery and preclinical trials.

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Generation of mice derived from embryonic stem cells using blastocysts of different developmental ages

Cited by 13 publications

References 17 publications

Acceptance of Embryonic Stem Cells by a Wide Developmental Range of Mouse Tetraploid Embryos1

Acceptance of Embryonic Stem Cells by a Wide Developmental Range of Mouse Tetraploid Embryos1

Improved Derivation Efficiency and Pluripotency of Stem Cells from the Refractory Inbred C57BL/6 Mouse Strain by Small Molecules

Genetically Engineered Mouse Models for Drug Development and Preclinical Trials

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