Strict control of telomerase activation using Cre-mediated inversion.
Strict control of telomerase activation using Cre-mediated inversion.
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DOI：
10.1186/1472-6750-6-10
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发表时间：
2006-02-20
影响因子：
3.5
通讯作者：
Harrington L
中科院分区：
文献类型：
--
作者：
Ungrin MD;Harrington L
人类细胞似乎对端粒酶逆转录酶hTERT的表达水平非常敏感。在不表达hTERT的原代细胞中，端粒随着每次连续的细胞分裂而腐蚀，最终导致端粒DNA的丢失，端粒DNA损伤反应的诱导，以及细胞衰老或危机的发生。在某些情况下，平均每个细胞少于一个适当剪接的hTERT转录物似乎足以恢复端粒酶活性和端粒维持，并克服有限的复制能力。为了强调这种敏感性，我们发现了一种广泛使用的包括蜕皮激素（muristerone）在内的转录诱导系统，即使在没有诱导的情况下，也能导致hTERT的充分表达，从而使人成纤维细胞永生。为了严格调控hTERT或任何其他感兴趣的基因的表达，我们开发了一种使用反平行loxP重组位点两侧的可逆表达盒的转录控制方法。当将hTERT cDNA引入人成纤维细胞时，端粒酶未被检测到活性，细胞群保持了致死表型。通过Cre重组酶的作用，将hTERT cDNA转化为转录能力取向后，细胞获得端粒酶活性，端粒DNA得到补充，种群得以永生。此外，利用荧光蛋白标记的表达，我们证明了在其他等基因细胞背景下，在活性和非活性状态之间重复反转特定转录本的能力。因此，这种二元表达系统提供了一种有用的遗传手段来严格调节给定基因的表达，或以互斥的方式控制至少两个不同基因的表达。
Human cells appear exquisitely sensitive to the levels of hTERT expression, the telomerase reverse transcriptase. In primary cells that do not express hTERT, telomeres erode with each successive cell division, leading to the eventual loss of telomere DNA, an induction of a telomere DNA damage response, and the onset of cellular senescence or crisis. In some instances, an average of less than one appropriately spliced hTERT transcript per cell appears sufficient to restore telomerase activity and telomere maintenance, and overcome finite replicative capacity. To underscore this sensitivity, we showed that a widely used system of transcriptional induction involving ecdysone (muristerone) led to sufficient expression of hTERT to immortalize human fibroblasts, even in the absence of induction. To permit tightly regulated expression of hTERT, or any other gene of interest, we developed a method of transcriptional control using an invertible expression cassette flanked by antiparallel loxP recombination sites. When introduced into human fibroblasts with the hTERT cDNA positioned in the opposite orientation relative to a constitutively active promoter, no telomerase activity was detected, and the cell population retained a mortal phenotype. Upon inversion of the hTERT cDNA to a transcriptionally competent orientation via the action of Cre recombinase, cells acquired telomerase activity, telomere DNA was replenished, and the population was immortalized. Further, using expression of a fluorescent protein marker, we demonstrated the ability to repeatedly invert specific transcripts between an active and inactive state in an otherwise isogenic cell background. This binary expression system thus provides a useful genetic means to strictly regulate the expression of a given gene, or to control the expression of at least two different genes in a mutually exclusive manner.
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影响因子：
3.7
作者：
MacKenzie, KL;Franco, S;Moore, MAS
通讯作者：
Moore, MAS
DOI：
10.1016/s0006-291x(03)01484-0
发表时间：
2003-09-05
影响因子：
3.1
作者：
Mondello, C;Chiesa, M;Nuzzo, F
通讯作者：
Nuzzo, F
影响因子：
56.9
作者：
FENG, JL;FUNK, WD;VILLEPONTEAU, B
通讯作者：
VILLEPONTEAU, B
影响因子：
9.2
作者：
Beattie, TL;Zhou, W;Harrington, L
通讯作者：
Harrington, L
影响因子：
56.9
作者：
Lingner, J;Hughes, TR;Cech, TR
通讯作者：
Cech, TR