Utilizing the LoxP-Stop-LoxP System to Control Transgenic ABC-Transporter Expression In Vitro.
Utilizing the LoxP-Stop-LoxP System to Control Transgenic ABC-Transporter Expression In Vitro.
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DOI：
10.3390/biom12050679
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发表时间：
2022-05-08
期刊：
影响因子：
5.5
通讯作者：
中科院分区：
文献类型：
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ABCA1和Abcg1是公认的促进胆固醇向载脂蛋白AI和高密度脂蛋白外流的两个ABC转运体。由于这两个ABC转运蛋白对胆固醇代谢至关重要，一些研究已经评估了ABCA1和Abcg1的表达通过ABC转运蛋白去除或过表达ABCA1/Abcg1对细胞胆固醇稳态的影响。然而，对于后者，目前还没有成熟的体外模型来有效地在各种培养细胞中诱导ABC转运蛋白的长期表达。因此，我们进行了体外原理验证研究，以确定loxP-Stop-loxP(LSL)系统是否能够提供Cre诱导的ABC转运蛋白表达。在我们的研究中，我们以ABCA1-LSL和Abcg1-LSL为基础，分别将ABCA1-LSL和Abcg1-LSL分别导入HEK293细胞及其衍生的293-CRE细胞。结果表明，在不表达ABCA1/Abcg1蛋白的HEK293细胞中，分别表达ABCA1-LSL和Abcg1-LSL的293-CRE细胞有ABCA1和Abcg1蛋白表达。当我们在293-Cre细胞中测量胆固醇外流时，我们观察到在高表达ABCA1的293-Cre细胞中ApoAI介导的胆固醇外流增强，在组成表达Abcg1的293-Cre细胞中HDL2介导的胆固醇外流。我们还观察到，在ABCA1过表达的293-Cre细胞中，HDL3介导的胆固醇外流显著增加，这表明ABCA1能够将胆固醇外流到小的高密度脂蛋白颗粒。我们的概念验证实验表明，LSL-系统可以在体外有效地调节ABC-转运蛋白的表达，这反过来又使得ABCA1/Abcg1-过表达的研究在细胞水平得到了广泛的研究。
ABCA1 and ABCG1 are two ABC-transporters well-recognized to promote the efflux of cholesterol to apoAI and HDL, respectively. As these two ABC-transporters are critical to cholesterol metabolism, several studies have assessed the impact of ABCA1 and ABCG1 expression on cellular cholesterol homeostasis through ABC-transporter ablation or overexpressing ABCA1/ABCG1. However, for the latter, there are currently no well-established in vitro models to effectively induce long-term ABC-transporter expression in a variety of cultured cells. Therefore, we performed proof-of-principle in vitro studies to determine whether a LoxP-Stop-LoxP (LSL) system would provide Cre-inducible ABC-transporter expression. In our studies, we transfected HEK293 cells and the HEK293-derived cell line 293-Cre cells with ABCA1-LSL and ABCG1-LSL-based plasmids. Our results showed that while the ABCA1/ABCG1 protein expression was absent in the transfected HEK293 cells, the ABCA1 and ABCG1 protein expression was detected in the 293-Cre cells transfected with ABCA1-LSL and ABCG1-LSL, respectively. When we measured cholesterol efflux in transfected 293-Cre cells, we observed an enhanced apoAI-mediated cholesterol efflux in 293-Cre cells overexpressing ABCA1, and an HDL2-mediated cholesterol efflux in 293-Cre cells constitutively expressing ABCG1. We also observed an appreciable increase in HDL3-mediated cholesterol efflux in ABCA1-overexpressing 293-Cre cells, which suggests that ABCA1 is capable of effluxing cholesterol to small HDL particles. Our proof-of-concept experiments demonstrate that the LSL-system can be used to effectively regulate ABC-transporter expression in vitro, which, in turn, allows ABCA1/ABCG1-overexpression to be extensively studied at the cellular level.
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影响因子：
1.9
作者：
Esobi IC;Barksdale C;Heard-Tate C;Reigers Powell R;Bruce TF;Stamatikos A
通讯作者：
Stamatikos A
影响因子：
14.9
作者：
Deprey, Kirsten;Batistatou, Nefeli;Kritzer, Joshua A.
通讯作者：
Kritzer, Joshua A.
影响因子：
2.7
作者：
Chong ZX;Yeap SK;Ho WY
通讯作者：
Ho WY
DOI：
10.1161/atvbaha.112.301110
发表时间：
2013-10
期刊：
Arteriosclerosis, thrombosis, and vascular biology
影响因子：
--
作者：
Bi X;Zhu X;Duong M;Boudyguina EY;Wilson MD;Gebre AK;Parks JS
通讯作者：
Parks JS
DOI：
10.1161/atvbaha.108.182303
发表时间：
2009-04-01
影响因子：
8.7
作者：
Brunham, Liam R.;Singaraja, Roshni R.;Hayden, Michael R.
通讯作者：
Hayden, Michael R.