Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Takahashi, Masanori; Makino, Sayaka; Kikkawa, Takako; Osumi, Noriko

doi:10.3791/51969

Cited by 19 publications

(15 citation statements)

References 28 publications

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“…At E8.5, pregnant females were euthanized by cervical dislocation and embryos were dissected for culture as described (Copp et al, ). RS for WEC was prepared and stored as described (Cockroft, ; Takahashi et al, ). Serum aliquots were thawed at 37 °C and filtered through a 0.45 μm filter (Merck) immediately before use.…”

Section: Methodsmentioning

confidence: 99%

“…Sera from humans, monkeys, cows and dogs were also tested, but none proved as effective as RS (Chambers, Klein, Nosel, Khairallah, & Romanow, ; Chatot, Klein, Piatek, & Pierro, ; Coelho, Weber, Klein, Daniels, & Hoagland, ; Flynn, Friedman, Black, & Klein, ). Hence, the use of RS as culture medium remains standard practice in WEC (Takahashi, Makino, Kikkawa, & Osumi, ).…”

Section: Introductionmentioning

confidence: 99%

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Mouse whole embryo culture: Evaluating the requirement for rat serum as culture medium

Culshaw

Savery

Greene

et al. 2019

Birth Defects Research

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Background Whole embryo culture is a valuable research method in mammalian developmental biology and birth defects research, enabling longitudinal studies of explanted organogenesis‐stage rodent embryos. Rat serum is the primary culture medium, and can sustain growth and development over limited periods as in utero. However, the cost, labor, and time to produce culture serum are factors limiting the uptake of the methodology. The goal of replacing or at least reducing rat serum usage in culture would be in accordance with the principles of “replacement, reduction, and refinement” of animals in research (the 3Rs). Methods We performed cultures of mouse embryos for 24 hr from embryonic day 8.5 in serum‐free media or in rat serum diluted with defined media, compared with 100% rat serum. Developmental parameters scored after culture included yolk sac circulation, dorsal axial length, somite number, protein content, and completion of cranial neural tube closure. Results A literature review revealed use of both serum‐free and diluted rat serum‐based media in whole embryo culture studies, but with almost no formal comparisons of culture success against 100% rat serum. Two serum‐free media were tested, but neither could sustain development as in 100% rat serum. Dilution of rat serum 1:1 with Glasgow Minimum Essential Medium plus defined supplements supported growth and development as well as whole rat serum, whereas other diluent media yielded substandard outcomes. Conclusion Rat serum usage cannot be avoided, to achieve high quality mouse embryo cultures, but rat usage can be reduced using medium containing diluted serum.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Mouse whole embryo culture: Evaluating the requirement for rat serum as culture medium

Culshaw

Savery

Greene

et al. 2019

Birth Defects Research

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“…Cultured rat embryos were electroporated as described previously (Takahashi, Makino, Kikkawa, & Osumi, ; Takahashi & Osumi, ). The plasmid vectors ( pCAX‐DsRed2, pCAX‐Pax6 and pFabp7‐EGFP s) were prepared at a concentration of 5 mg/ml and microinjected into the rhombencephalic, mesencephalic and telencephalic vesicles at E11.5.…”

Section: Methodsmentioning

confidence: 99%

Pax6‐dependent regulation of the rat Fabp7 promoter activity

et al. 2018

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Fabp7 gene encodes a brain-specific fatty acid-binding protein that is widely used as a marker for neural stem cells. Here, we report that the activity of rat Fabp7 promoter was regulated directly by a transcription factor, Pax6. Deletion analyses identified an essential region (-837 to -64 from transcription start site) in the rat Fabp7 promoter. This region controls promoter activity in rat embryos and in the mouse cultured cell line MEB5. Over-expressing wild-type Pax6 or a dominant-negative Pax6 mutant enhanced and suppressed, respectively, the promoter activity. Pax6 can bind the region directly, although the region contains no clear binding motif for Pax6. The rat Fabp7 promoter also contains conserved binding sites for Pbx/POU (-384 to -377) and CBF1 (-270 to -262). However, specific deletion of the sites showed no significant reduction in the promoter activity, although a gel mobility shift assay confirmed that CBF1 binds the conserved sequence. Taken together, these results suggest that the rat Fabp7 promoter is mainly regulated by Pax6. The Pax6-dependent regulation of the rat Fabp7 expression might have an evolutionary aspect between rat and mouse; the former may need to efficiently use fatty acids to make the brain bigger than the latter.

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“…Appropriate matings were performed and detection of copulatory plugs was designated embryonic day (E) 0.5. Retired breeder rats were used for serum extraction (Takahashi et al, 2014) …”

Section: Mice and Ratsmentioning

confidence: 99%

Bmp signaling maintains a mesoderm progenitor cell state in the mouse tailbud

Sharma¹,

Shafer²,

Bareke³

et al. 2017

Journal of Cell Science

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Caudal somites are generated from a pool of progenitor cells located in the tailbud region. These progenitor cells form the presomitic mesoderm that gradually differentiates into somites under the action of the segmentation clock. The signals responsible for tailbud mesoderm progenitor pool maintenance during axial elongation are still elusive. Here, we show that Bmp signaling is sufficient to activate the entire mesoderm progenitor gene signature in primary cultures of caudal mesoderm cells. Bmp signaling acts through the key regulatory genes brachyury (T ) and Nkx1-2 and contributes to the activation of several other regulators of the mesoderm progenitor gene network. In the absence of Bmp signaling, tailbud mesoderm progenitor cells acquire aberrant gene expression signatures of the heart, blood, muscle and skeletal embryonic lineages. Treatment of embryos with the Bmp inhibitor noggin confirmed the requirement for Bmp signaling for normal T expression and the prevention of abnormal lineage marker activation. Together, these results identify Bmp signaling as a non-cell-autonomous signal necessary for mesoderm progenitor cell homeostasis.

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Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Cited by 19 publications

References 28 publications

Mouse whole embryo culture: Evaluating the requirement for rat serum as culture medium

Mouse whole embryo culture: Evaluating the requirement for rat serum as culture medium

Pax6‐dependent regulation of the rat Fabp7 promoter activity

Bmp signaling maintains a mesoderm progenitor cell state in the mouse tailbud

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