| Fluorescence Measurement and Calibration of Intracellular pH in Starfish Oocytes
|
|
Author:
Date:
2020-10-05
[Abstract] Oocyte maturation is a process wherein an oocyte arrested at prophase of meiosis I resumes meiosis to become a fertilizable egg. In starfish ovaries, a hormone released from follicle cells activates the oocytes, resulting in an increase in their intracellular pH (pHi), which is required for spindle assembly. Herein, we describe a protocol for pHi measurement in living oocytes microinjected with the pH-sensitive dye BCECF. For in vivo BCECF calibration, we treated oocytes with artificial seawater containing CH3COONH4 to clamp pHi, injected pH-standard solutions, and converted the BCECF fluorescence intensity ratios to pHi values. Of note, if the actual pHi is higher or lower than the known pH of injected ...
[摘要] [摘要]卵母细胞成熟是在减数分裂I前期停滞的卵母细胞恢复减数分裂成为可受精卵的过程。在海星卵巢中，从卵泡细胞释放的激素激活卵母细胞，导致其胞内pH（pH i ）升高，这是纺锤体组装所必需的。在本文中，我们描述了在用pH敏感染料BCECF显微注射的活卵母细胞中测量pH i的方案。对于在体内BCECF校准，我们用含有CH人造海水的卵母细胞3 COONH 4到夹具的pH我 ，注入pH标准溶液，并将BCECF荧光强度比值转换为pH i值。值得注意的是，如果实际pH值我比注入标准溶液的pH值已知更高或更低时，BCECF的荧光强度比将分别减小或增大。另一方面，注入溶液的pH值没有显示出荧光强度的变化，应该认为是实际pH值i 。这些用于pH i校准和钳制的方法简单且可重现。
关键词：细胞内pH，BCECF ，卵母细胞，显微注射，海星，标定
[背景]细胞内pH （pH为我）测量为细胞生物学的研究中是非常有用的方法，因为pH值我起着在多种细胞过程中起重要作用，如配子活化（约翰逊和EPEL ，1976; Shen和斯坦哈特，1978 ;蒂尔尼等，1978 ），细胞分裂（Schuldiner和Rozengurt ，1982; Moolenaar译者等，1983;阿南德和普拉萨德，1989; ...
|
|
|
| Gene Expression and Genome Editing Systems by Direct Delivery of Macromolecules Into Rice Egg Cells and Zygotes
|
|
Author:
Date:
2020-07-20
[Abstract] Polyethylene glycol calcium (PEG-Ca2+)-mediated transfection allows rapid and efficient examination to analyze diverse cellular functions of genes of interest. In plant cells, macromolecules, such as DNA, RNA and protein, are delivered into protoplasts derived from somatic tissues or calli via PEG-Ca2+ transfection. To broaden and develop the scope of investigations using plant gametes and zygotes, a procedure for direct delivery of macromolecules into these cells has recently been established using PEG-Ca2+ transfection. This PEG-Ca2+-mediated delivery into rice egg cells/zygotes consists of four microtechniques, (i) isolation of gametes, (ii) production of zygotes by electrofusion of gametes, (iii) PEG-Ca2+-mediated delivery of ...
[摘要] [摘要 ] P olyethylene乙二醇钙（PEG-的Ca 2+ ）介导的转染允许快速和有效的检查来分析感兴趣的基因的不同的细胞功能。在植物细胞中，大分子，例如DNA，RNA和蛋白质，通过PEG-Ca 2+ 转染被传递到源自体组织或愈伤组织的原生质体中。牛逼Ø拓宽和开发利用植物的配子和合子，直接递送大分子进入这些细胞最近已经采用PEG-钙建立过程调查范围2+ 转染。这种PEG-Ca 2+ 介导的传递至水稻卵细胞/受精卵的过程包括四个 微技术，（i ）配子的分离，（ii）通过配子的电融合产生合子，（iii）PEG-Ca 2+ 介导的大分子向分离的卵细胞或产生的合子的传递，以及（iv）的培养和随后的分析转染的卵细胞/受精卵。因为对于完整的协议microtechniques （我）和（ii）已在户报告等人。，2016年，microtechniques （iii）和（iv）的主要是在第描述是协议。
[背景 ] 在被子植物中，受精和随后的发育事件，诸如胚胎发生和胚乳发育，发生在深深嵌入卵形组织胚囊（Nawaschin ，1898; Guignard ，1899;罗素，1992;拉加，2003） ...
|
|
|