Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis.

Naive pluripotency is manifest in the preimplantation mammalian embryo. Here we determine transcriptome dynamics of mouse development from the eight-cell stage to postimplantation using lineage-specific RNA sequencing. This method combines high sensitivity and reporter-based fate assignment to acquire the full spectrum of gene expression from discrete embryonic cell types. We define expression modules indicative of developmental state and temporal regulatory patterns marking the establishment and dissolution of naive pluripotency in vivo. Analysis of embryonic stem cells and diapaused embryos reveals near-complete conservation of the core transcriptional circuitry operative in the preimplantation epiblast. Comparison to inner cell masses of marmoset primate blastocysts identifies a similar complement of pluripotency factors but use of alternative signaling pathways. Embryo culture experiments further indicate that marmoset embryos utilize WNT signaling during early lineage segregation, unlike rodents. These findings support a conserved transcription factor foundation for naive pluripotency while revealing species-specific regulatory features of lineage segregation.We thank Peter Humphreys for assistance with imaging, and Samuel Jameson and staff for mouse husbandry. We are grateful to Charis Drummer, Ayako Sedohara, Akiko Shimada, Yuko Yamada, Ryo Oiwa, and Takeshi Kuge for technical support with marmoset embryo recovery. Illumina sequencing was provided by Bettina Haase and Dinko Pavlinic at the EMBL Genomics Core Facility. This work was supported by funding from the Wellcome Trust, the Genome Biology Unit of the European Molecular Biology Laboratory, BBSRC grants BB/G015678/1 and BB/M004023/1, an MRC Centenary Award, and the Louis Jeantet Foundation. A.S. is a Medical Research Council Professor.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.devcel.2015.10.01

Generation of transgenic cynomolgus monkeys that express green fluorescent protein throughout the whole body.

Nonhuman primates are valuable for human disease modelling, because rodents poorly recapitulate some human diseases such as Parkinson\u27s disease and Alzheimer\u27s disease amongst others. Here, we report for the first time, the generation of green fluorescent protein (GFP) transgenic cynomolgus monkeys by lentivirus infection. Our data show that the use of a human cytomegalovirus immediate-early enhancer and chicken beta actin promoter (CAG) directed the ubiquitous expression of the transgene in cynomolgus monkeys. We also found that injection into mature oocytes before fertilization achieved homogenous expression of GFP in each tissue, including the amnion, and fibroblasts, whereas injection into fertilized oocytes generated a transgenic cynomolgus monkey with mosaic GFP expression. Thus, the injection timing was important to create transgenic cynomolgus monkeys that expressed GFP homogenously in each of the various tissues. The strategy established in this work will be useful for the generation of transgenic cynomolgus monkeys for transplantation studies as well as biomedical research

Establishing cynomolgus monkey transplant models using tailor-made ES cells

科学研究費補助金研究成果報告書研究種目: 基盤研究(B)研究期間: 2006～2009課題番号: 18300140研究代表者: 鳥居 隆三（滋賀医科大学・動物生命科学研究センター・教授）研究分担者: 高田 達之（滋賀医科大学・動物生命科学研究センター・准教授）研究分担者: 土屋 英明（滋賀医科大学・動物生命科学研究センター・技術専門職員）研究分担者: 岡原（成田） 純子（滋賀医科大学・動物生命科学研究センター・助教）研究協力者: 岩谷 千鶴（滋賀医科大学・動物生命科学研究センター・教務補佐員）研究協力者: 山崎 樹里（滋賀医科大学・動物生命科学研究センター・教務補佐員

Devices for nonsurgical embryo transfer.

<p>Newly developed devices for nonsurgical embryo transfer. (A) I: 23-G, 120-mm-long blunt-end stainless steel stylet II: polyethylene 160-mm-long cannula (inner diameter 0.28 mm, outer diameter 0.61 mm) for embryo transfer, III: Fluon ETFE 20-G, 108-mm-long cannula (A blunt/tapered cannula; inner diameter 0.8 mm, outer diameter 1.10 mm), IV: endoscope for small animals, V: tapered to 4.2 mm, 6.0 mm in diameter and tapered to 5.0 mm, 7.0 mm in diameter and tapered to 5.3 mm at one end of the glass tubes for vaginal dilation and manipulation of the cannulae. (B) Scheme for nonsurgical embryo transfer.</p

Pregnancy and birth rates following nonsurgical embryo transfer to recipients of common marmoset embryos produced by ICSI.

<p>Values within the same column with different letters (a, b) differ significantly (p<0.05), χ²-test.</p><p>*¹Numbers in parentheses were calculated from total recipients.</p><p>*²Numbers in parentheses were calculated from total embryos.</p

Effect of fertilization method on <i>in vitro</i> development of marmoset oocytes.

<p>Values within the same column with different letters (a, b) differ significantly (p<0.05), χ²-test.</p><p>*¹Numbers in parentheses were calculated from total oocytes.</p><p>*²Numbers in parentheses were calculated from fertilized oocytes.</p

The length of embryonic development until the blastocyst stage of the embryos derived by ICSI and IVF.

<p>*Numbers in parentheses were calculated from total blastocysts.</p><p>Day 0: Day of fertilization.</p

First offspring produced by ICSI in marmosets.

<p>Four healthy neonates were generated using ICSI. A female (A) 635 produced by blastocyst transfer and a male neonate (B) 732, male (C) 737 and female neonate (D) 640 from 6-cell- to 8-cell stage embryo transfer.</p