生物谷報(bào)道：體細(xì)胞核移植通常采用電融合法和直接注射法,。電融合法是用去核管（外徑25-30µm）注入到去核卵母細(xì)胞質(zhì)透明帶下即卵周隙，但要保證細(xì)胞膜的完整,，才能使供體細(xì)胞膜與卵母細(xì)胞質(zhì)膜粘合在一起,，有利于融合,。最后通過電融合，使得二者合二為一,，當(dāng)然最后仍然需要激活才可以使得重組胚發(fā)育,。
而直接注核法，通常是用較細(xì)的注核針（外徑10-15µm）反復(fù)抽吸供體細(xì)胞使其質(zhì)膜破裂,，然后直接注入到去核的卵母細(xì)胞胞質(zhì)中,。這里的供核要求質(zhì)膜破裂，有助于釋放核進(jìn)行再程序化,。注核之后仍然需要激活,，使重組胚進(jìn)行隨后的發(fā)育。
目前在豬的體細(xì)胞核移植上,，文獻(xiàn)上大多采用的是直接注射法,。而本文與同不同的地方就是采用直接注核,，將一個(gè)完整的體細(xì)胞（供核）直接注入到胞質(zhì)中，然后通過染色,，發(fā)現(xiàn)成纖維細(xì)胞的核膜在注射后6小時(shí),，膜已經(jīng)破裂了，從而進(jìn)行隨后一系列的發(fā)育,，得到了克隆豬仔,。文中給出精美圖片，很有說服力,。
生物谷專家認(rèn)為,，文中給出的可能的解釋有二：一是卵母細(xì)胞積極地識(shí)別了供核細(xì)胞的質(zhì)膜或者是其表面的附屬蛋白，而這種識(shí)別將導(dǎo)致卵母細(xì)胞很快降解供核細(xì)胞的質(zhì)膜或者將之運(yùn)輸?shù)铰涯讣?xì)胞的表面,，這種活躍的分解作用隨之將釋放核進(jìn)行再程序化,。二是在整個(gè)供核注入到胞質(zhì)過程中，機(jī)械力量可能將供核細(xì)胞膜弄破了,，那么破漏質(zhì)膜將釋放核,，造成核的再程序化。
事實(shí)上,，很多文獻(xiàn)報(bào)道,，直接注射法比融合法構(gòu)建重組胚的效率高。Trounson等（1998）等發(fā)現(xiàn),，牛體細(xì)胞的直接注核法是克隆牛胚胎的一種相對(duì)有效的途徑,。同樣用直接注射法也得到了體細(xì)胞核移植小鼠（Wakayama et al., 1999; Baguisi & Overstrom, 2000）、豬（Onishi et al., 2000）,、山羊（郭繼彤,，2000）。研究發(fā)現(xiàn),，由于體細(xì)胞與卵母細(xì)胞的體積相差很大,，相容性也差，電融合構(gòu)建胚胎比較困難（Kubota et al., 2000）,，遠(yuǎn)低于直接注射法構(gòu)建的效率（Trounson et al., 1998）,，所以直接注射法在體細(xì)胞核移植研究中有被廣泛應(yīng)用的趨勢(shì)。而本文的研究又給出了一個(gè)很有利的證明,。
FIG. 1. The whole-cell injection procedure and cloned embryos. A) A fibroblast cell (arrowhead) is aspirated into the injection pipette. B) The cell is expelled into the cytoplasm of the enucleated oocyte. C) Verification of the absence of the donor cell in the injection pipette. D) Hatched blastocysts (Day 6) produced by whole-cell injection of fibroblast cells. Original magnification x100
FIG. 2. Nuclear events following whole-cell injection. A) Plasma membrane of fibroblast cells was stained with live-membrane fluorescent dye (green). The injected cells were visible in the oocytes' cytoplasm following injection. B) The fibroblast nucleus (arrowhead) stained with Hoechst 33342 (blue) was observed 6 h following cell injection at which time the plasma membrane of the donor cell was no longer visible. C) Full nucleus swelling (arrowhead) was observed 12 h after oocyte activation. D) Hatched blastocysts injected with fibroblast cells. E) A hatched blastocyst stained with Hoechst 33342. Original magnification: A), x75; B/E), x200; C/D), x150
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