Hydrodynamic delivery of Cre protein to lineage-mark or time-stamp mouse hepatocytes in situ.
Hydrodynamic delivery of Cre protein to lineage-mark or time-stamp mouse hepatocytes in situ.
复制标题
DOI：
10.1371/journal.pone.0091219
复制
发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Schmidt EE
中科院分区：
文献类型：
--
作者：
Sonsteng KM;Prigge JR;Talago EA;June RK;Schmidt EE
Cre 响应荧光标记等位基因是小鼠细胞谱系追踪的强大工具；然而，它们的效用受到 Cre 活动监管的限制。当靶向肝细胞时，Cre表达质粒的流体动力学递送可以转化Cre反应等位基因，而不诱导通常与病毒衍生的Cre表达载体相关的细胞内或全身抗病毒反应。在该方法中，快速大容量静脉接种诱导肝细胞靶向摄取细胞外分子。在这里，我们测试了 Cre 蛋白或与 HIV-TAT 细胞穿透肽融合的 Cre 的流体动力学递送是否可以转化小鼠肝细胞中的 Cre 反应报告基因。 2 nmol Cre 或 TAT-Cre 蛋白的流体动力学递送在 5% 至 20% 的肝细胞中转化了报告等位基因。这两种蛋白质在内皮细胞、非肝器官或肝脏中的非肝细胞中均未产生可检测到的 Cre 活性。使用 Cre 响应标记纯合子小鼠，将红色（Cre-naïve）或绿色（Cre-converted）荧光蛋白引导至细胞核，我们评估了亚饱和 Cre 活性。流体动力学接种 Cre 蛋白一个月后，58% 的绿色肝细胞核也变成红色，表明获得足够 Cre 将一个标记等位基因转化为绿色的肝细胞中不到一半能够转化所有等位基因。为了进行比较，在流体动力传递具有弱启动子的 Cre 表达质粒一个月后，只有 26% 的绿色细胞核也呈红色。我们的结果表明，Cre 蛋白的流体动力学递送允许肝细胞中的快速等位基因转化，但 Cre 活性是亚饱和的，因此许多细胞不会转化多个 Cre 响应等位基因。
Cre-responsive fluorescent marker alleles are powerful tools for cell lineage tracing in mice; however their utility is limited by regulation of Cre activity. When targeting hepatocytes, hydrodynamic delivery of a Cre-expression plasmid can convert Cre-responsive alleles without inducing the intracellular or systemic antiviral responses often associated with viral-derived Cre-expression vectors. In this method, rapid high-volume intravenous inoculation induces hepatocyte-targeted uptake of extracellular molecules. Here we tested whether hydrodynamic delivery of Cre protein or Cre fused to the HIV-TAT cell-penetrating peptide could convert Cre-responsive reporters in hepatocytes of mice. Hydrodynamic delivery of 2 nmol of either Cre or TAT-Cre protein converted the reporter allele in 5 to 20% of hepatocytes. Neither protein gave detectable Cre activity in endothelia, non-liver organs, or non-hepatocyte cells in liver. Using mice homozygous for a Cre-responsive marker that directs red- (Cre-naïve) or green- (Cre-converted) fluorescent proteins to the nucleus, we assessed sub-saturation Cre-activity. One month after hydrodynamic inoculation with Cre protein, 58% of hepatocyte nuclei that were green were also red, indicating that less than half of the hepatocytes that had obtained enough Cre to convert one marker allele to green were able to convert all alleles. For comparison, one month after hydrodynamic delivery of a Cre-expression plasmid with a weak promoter, only 26% of the green nuclei were also red. Our results show that hydrodynamic delivery of Cre protein allows rapid allelic conversion in hepatocytes, but Cre-activity is sub-saturating so many cells will not convert multiple Cre-responsive alleles.
登录
查看更多内容
影响因子：
4.2
作者：
Muruve, DA;Barnes, MJ;Libermann, TA
通讯作者：
Libermann, TA
影响因子：
13.5
作者：
Huang, Mei;Sun, Rui;Tian, Zhigang
通讯作者：
Tian, Zhigang
影响因子：
1.5
作者：
Brockschniedier, Damian;Pechmann, Yvonne;Riethmacher, Dieter
通讯作者：
Riethmacher, Dieter
影响因子：
2.5
作者：
Prigge, Justin R.;Wiley, James A.;Schmidt, Edward E.
通讯作者：
Schmidt, Edward E.
影响因子：
4.2
作者：
Christ, M;Louis, B;Mehtali, M
通讯作者：
Mehtali, M