Viral transduction of primary Schwann cells using a Cre-lox system to regulate GDNF expression.
Viral transduction of primary Schwann cells using a Cre-lox system to regulate GDNF expression.
复制标题
DOI：
10.1002/bit.25247
复制
发表时间：
2014-09
影响因子：
3.8
通讯作者：
Wood, Matthew D.
中科院分区：
文献类型：
--
作者：
Wu-Fienberg, Yuewei;Moore, Amy M.;Marquardt, Laura M.;Newton, Piyaraj;Johnson, Philip J.;Mackinnon, Susan E.;Sakiyama-Elbert, Shelly E.;Wood, Matthew D.
关键词：
胶质细胞源性神经营养因子(GDNF)是一种高效的神经营养因子，可促进运动神经的再生。然而，最近的研究已经确定，延长GDNF对再生轴突的输送可以将运动轴突困在GDNF输送的位置。这种缠绕导致可用于重新支配肌肉的运动神经轴突减少。为了解决这个问题，我们设计了一个基于细胞的GDNF表达系统，该系统可以使用可诱导的基因切除机制来暂时调节蛋白质的表达，以防止表达部位的捕获。为了设计这一调控GDNF表达的系统，我们将两个慢病毒载体导入雪旺细胞(Schwann cell，SCs)，其中一个载体含有一个结构性活性的GDNF转基因基因，两侧有两个loxP位点，另一个载体含有四环素可诱导的cre转基因基因及其结构性活性反式激活因子。这些干细胞过度表达GDNF，但可通过给予四环素家族抗生素，如强力霉素抑制其表达。通过酶联免疫吸附试验(ELISA)检测，与未转导的对照组相比，工程化的SCs产生了明显更多的GDNF。经多西环素治疗后，与未治疗的干细胞相比，这些干细胞产生的GDNF水平显著降低，并且诱导的轴突延伸更少。免疫组织化学(IHC)显示经多西环素处理的工程化SCs的Cre重组酶表达显著增加，这为观察到GDNF表达水平和生物活性变化的机制提供了证据。这种基于细胞的GDNF表达系统有可能在未来的体内研究中提供一个时间控制的GDNF来源来促进轴突生长。
Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor known to enhance motor nerve regeneration following its delivery. However, recent studies have determined that extended GDNF delivery to regenerating axons can entrap motor axons at the site of GDNF delivery. This entrapment leads to reduced motor axons available to reinnervate muscle. To address this issue, we designed a cell-based GDNF expression system that can temporally regulate protein expression using an inducible gene excision mechanism to prevent entrapment at the site of expression. To design this system for regulation of GDNF expression, we transduced two lentiviral vectors, one containing a constitutively active GDNF transgene flanked by two loxP sites, and the other containing a tetracycline-inducible cre transgene along with its constitutively active transactivator, into Schwann cells (SCs). These SCs over-express GDNF, but expression can be suppressed through the administration of tetracycline family antibiotics, such as doxycycline. The engineered SCs produced significantly more GDNF as compared to untransduced controls, as measured by enzyme-linked immunosorbent assay (ELISA). Following doxycycline treatment, these SCs produced significantly lower levels of GDNF and induced less neurite extension as compared to untreated SCs. Engineered SCs treated with doxycycline showed a marked increase in Cre recombinase expression, as visualized by immunohistochemistry (IHC), providing evidence of a mechanism for the observed changes in GDNF expression levels and biological activity. This cell-based GDNF expression system could have potential for future in vivo studies to provide a temporally-controlled GDNF source to promote axon growth.
登录
查看更多内容
影响因子：
1.7
作者：
Gordon, Tessa
通讯作者：
Gordon, Tessa
影响因子：
2.3
作者：
Boyd, Kirsty U.;Nimigan, Andre S.;Mackinnon, Susan E.
通讯作者：
Mackinnon, Susan E.
影响因子：
14.8
作者：
Kaewkhaw, Rossukon;Scutt, Andy M.;Haycock, John W.
通讯作者：
Haycock, John W.
影响因子：
5.3
作者：
LEVI, ADO;GUENARD, LV;BUNGE, RP
通讯作者：
BUNGE, RP
影响因子：
3.7
作者：
Eggers R;de Winter F;Hoyng SA;Roet KC;Ehlert EM;Malessy MJ;Verhaagen J;Tannemaat MR
通讯作者：
Tannemaat MR