An in vitro method for analysis of chondrogenesis in limb mesenchyme from individual transgenic (hdf) embryos.
An in vitro method for analysis of chondrogenesis in limb mesenchyme from individual transgenic (hdf) embryos.
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一种用于分析个体转基因 (hdf) 胚胎肢体间充质软骨形成的体外方法。
DOI：
10.1007/s11022-004-9803-3
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Capehart,AnthonyA
中科院分区：
文献类型：
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作者：
Gillotte,DanielleM;Fox,PatriciaL;Mjaatvedt,CoreyH;Hoffman,Stanley;Capehart,AnthonyA
本研究描述了一种简单、快速的方案，用于培养来自个体交配后 10.5 天的小鼠胚胎的肢体组织，该方案支持在六天内的软骨分化。该技术与其他常用的利用混合肢体组织的方法的不同之处在于：1）使用来自单个胚胎的前肢作为供体组织； 2)通过非常温和的酶消化（0.1%胰蛋白酶，5分钟）解离肢体组织； 3) 将细胞悬液以较低密度（1 × 107 与 2 × 107 细胞/ml）涂板，体积减少至 3-5 μl。在这些改进的条件下，为了增加每个胚胎的肢体细胞产量，组织化学和免疫组织化学分析证明了前软骨聚集体的可重复形成以及随后在可预测的时间过程中明显的软骨形成。使用这种培养方案，对来自杂合子胚胎的肢体间充质进行分析，该杂合子胚胎带有编码硫酸软骨素蛋白多糖核心蛋白Versacan的Cspg2基因的插入突变，揭示了与野生型同窝动物观察到的总体相似的软骨形成潜力。这项技术很容易在体外培养来自该发育阶段的个体小鼠胚胎的肢芽间质，并且研究人员可以利用该技术来研究 hdf 和其他转基因突变对哺乳动物肢体体外发育的影响。
The present study describes a simple, rapid protocol for culture for limb tissue from individual 10.5-day post coitum mouse embryos that supports cartilage differentiation over a six-day period. This technique differs from other commonly used methods utilizing pooled limb tissue in that: 1) forelimbs from individual embryos were used as donor tissue; 2) limb tissue was dissociated by very gentle enzymatic digest (0.1% trypsin, 5 min); and, 3) cell suspensions were plated at a lower density (1 × 107vs. 2 × 107cells/ml) in a reduced volume of 3–5 μl. Under these modified conditions to increase limb cell yield from each embryo, histochemical and immunohistochemical analyses demonstrated reproducible for-mation of precartilage aggregates and subsequent overt chondrogenesis over a predictable time course. Using this culture protocol, analysis of limb mesenchyme from heterozygoushdfembryos, which bear an insertional mutation of theCspg2gene encoding the core protein of the chondroitin sulfate proteoglycan, versican, revealed an overall similar chondrogenic potential to that observed for wild-type littermates. This technique readily enables in vitro culture of limb bud mesenchyme from individual mouse embryos at this developmental stage and may be utilized by investigators to study the effects of thehdfand other transgenic mutations on mammalian limb developmentin vitro.