Tetracycline-inducible gene expression in conditionally immortalized mouse podocytes.
Tetracycline-inducible gene expression in conditionally immortalized mouse podocytes.
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DOI：
10.1159/000151770
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发表时间：
2009
影响因子：
4.2
通讯作者：
Kopp JB
中科院分区：
文献类型：
--
作者：
Kajiyama H;Titus S;Austin CP;Chiotos K;Matsumoto T;Sakairi T;Kopp JB
连续永生化足细胞是有价值的研究工具，但难以有效地转染并且不能提供分级的转基因表达。采用四环素诱导系统建立了永生化小鼠足细胞系。克隆了从携带两种转基因的转基因小鼠中分离的肾小球细胞，所述两种转基因是NPHS 2-反向四环素控制的反式激活因子rtTA（A转基因）和H2-Kb-热敏SV 40 T，ts 58 A（I转基因）。用pBI-EGFP（增强型绿色荧光蛋白，G转基因）和分别用ptTS-Neo（转录抑制因子，T转基因）转染表达WT 1和突触足蛋白的一个克隆（AI足细胞），以产生稳定的转化体、AIG足细胞和AIT足细胞。强力霉素处理后，AIG足细胞在33和37°C下表达EGFP，并保留podocin和rtTA mRNA表达以及温度敏感性生长调节。AIT足细胞，瞬时转染的荧光素酶-BI-EGFP（LG转基因），显示减少的背景表达的EGFP和荧光素酶的情况下，强力霉素。在通过用LG转基因稳定转染AIT足细胞产生的AITLG足细胞中，在33 ° C和37°C下，多西环素以时间和浓度依赖性方式严格调节荧光素酶表达，尽管背景表达没有完全消除。这些足细胞保留了温度敏感的生长调节和足细胞分化标志物的表达。小鼠足细胞有效表达四环素诱导的转基因，同时保留分化标记。
Conditionally immortalized podocytes are valuable research tools but are difficult to efficiently transfect and do not provide graded transgene expression. Conditionally immortalized mouse podocyte cell lines were established employing a tetracycline-inducible system. Glomerular cells, isolated from transgenic mice bearing two transgenes, NPHS2-reverse tetracycline-controlled transactivator, rtTA (A transgene) and H2-Kb-thermosensitive SV40 T, ts58A (I transgene), were cloned. One clone (AI podocytes) expressing WT1 and synaptopodin was transfected with pBI-EGFP (enhanced green fluorescent protein, G transgene) and separately with ptTS-Neo (transcriptional suppressor, T transgene) to produce stable transformants, AIG podocytes and AIT podocytes. AIG podocytes expressed EGFP at 33 and 37°C after doxycycline treatment, and retained podocin and rtTA mRNA expression and temperature-sensitive growth regulation. AIT podocytes, transiently transfected with luciferase-BI-EGFP (LG transgene), showed reduced background expression of EGFP and luciferase in the absence of doxycycline. In AITLG podocytes, generated by stable transfection of AIT podocytes with the LG transgene, luciferase expression was tightly regulated by doxycycline in a time- and concentration-dependent manner both at 33 and 37°C, although background expression was not entirely eliminated. These podocytes retained temperature-sensitive growth regulation and expression of podocyte differentiation markers. Mouse podocytes expressed tetracycline-induced transgenes efficiently while retaining differentiation markers.
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影响因子：
3.2
作者：
Fan, QF;Ding, J;Deng, JH
通讯作者：
Deng, JH
DOI：
10.1073/pnas.88.12.5096
发表时间：
1991-06-01
影响因子：
11.1
作者：
JAT, PS;NOBLE, MD;KIOUSSIS, D
通讯作者：
KIOUSSIS, D
DOI：
10.1073/pnas.130192197
发表时间：
2000-07-05
影响因子：
11.1
作者：
Urlinger, S;Baron, U;Hillen, W
通讯作者：
Hillen, W
影响因子：
3.7
作者：
Mundel, P;Reiser, J;Zeller, R
通讯作者：
Zeller, R
影响因子：
13.6
作者：
Saleem, MA;O'Hare, MJ;Mundel, P
通讯作者：
Mundel, P