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发表于 2022-7-29 00:03:15
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研究为什么0代的病毒，插入内含子的克隆和不插入内含子的克隆有区别。和基因组RNA的正确剪切有关。
对细胞核和胞浆中的病毒基因组进行分析。
the
extent of splicing of the genomic RNA, the proportion of viral
RNA molecules with the native (spliced) sequence accumulated in the cytoplasm of BHK-pAPN cells transfected with
these plasmids was analyzed.
分离胞浆和胞核：
转染24/40小时后的细胞用含0.5% NP-40的缓冲液进行裂解。
离心后，
上清加入含8M尿素-1.5%SDS的缓冲液。胞浆RNA通过酚-氯仿抽提。
沉淀用0.25M蔗糖-1.5%柠檬酸钠重悬
buffer at 20 and 40 h posttransfection. After centrifugation, the
supernatants were treated with a buffer containing 8 M urea–
1.5% sodium dodecyl sulfate, and then cytoplasmic RNA was
extracted with phenol-chloroform, while the pellets were resuspended in 0.25 M sucrose–1.5% (wt/vol) sodium citrate and
spun through a 0.88 M sucrose–1.5% (wt/vol) sodium citrate
layer to purify nuclei (8). This procedure yields nuclei with a
purity of over 95% as determined by optical microscopy. The
nuclear RNA was obtained after phenol-chloroform extraction
and ethanol precipitation.
Both cytoplasmic and nuclear RNA
were treated with RNase-free DNase I to remove contaminating plasmid DNA (5). A viral RNA fragment that included the
two positions of insertion of the intron (from nt 9291 to 9758)
was amplified by RT-PCR. PCR products were analyzed on a
2% agarose gel, and the intensities of the bands corresponding
to amplification of spliced and nonspliced RNAs for each situation were estimated by using a gel documentation system
(model 2000; Bio-Rad).
These bands represent the amount of
each RNA accumulated in both cell compartments. It should
be noted that the amount of RNA accumulated in the cytoplasm corresponds to the viral RNA spliced in the nucleus and translocated to the cytoplasm plus the newly synthesized RNA
produced in the cytoplasm by the viral replicase. About 10 to
15% of total genomic RNA molecules accumulated in the cells
corresponded to the spliced form, when the intron had been
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