AAV8 Ins1-Cre can produce efficient β-cell recombination but requires consideration of off-target effects
AAV8 Ins1-Cre can produce efficient β-cell recombination but requires consideration of off-target effects
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DOI：
10.1038/s41598-020-67136-w
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发表时间：
2020-06-29
影响因子：
4.6
通讯作者：
Kieffer, Timothy J.
中科院分区：
文献类型：
--
作者：
Ramzy, Adam;Tuduri, Eva;Kieffer, Timothy J.
体内遗传操作用于研究基因缺失或重新表达对β细胞功能和生物体生理的影响。Cre-LoxP是一种由Cre重组酶识别基因两侧的LoxP位点的系统。Cre转基因小鼠是用于传递Cre的最流行的技术，但许多模型存在脱靶重组、细胞功能受损和动物生产成本高的缺点。诱导型雌激素受体偶联Cre模型面临他莫昔芬的漏性重组和混杂效应。作为替代方案，我们描述了一种腺相关病毒（AAV），它具有大鼠胰岛素1启动子驱动Cre重组酶（AAV8 Ins1-Cre），该病毒经济且快速，并且具有有限的警告。腹腔注射AAV8 Ins1-Cre可产生有效的β -细胞重组，并伴有肝脏、外分泌胰腺、α -细胞、δ -细胞和下丘脑的重组。通过胰管给药的低剂量保留了良好的细胞重组率和有限的脱靶重组率。与转基因小鼠的诱导Cre不同，AAV8 Ins1-Cre不需要他莫昔芬，避免了过早重组。我们通过诱导胰岛素敲除小鼠(Ins1（-/-);Ins2(f/f)）诱导高血糖，证明了该技术的实用性。aav介导的Cre在β细胞中的表达为诱导敲除研究提供了一种有效的替代转基因方法。
In vivo genetic manipulation is used to study the impact of gene deletion or re-expression on beta -cell function and organism physiology. Cre-LoxP is a system wherein LoxP sites flanking a gene are recognized by Cre recombinase. Cre transgenic mice are the most prevalent technology used to deliver Cre but many models have caveats of off-target recombination, impaired beta -cell function, and high cost of animal production. Inducible estrogen receptor conjugated Cre models face leaky recombination and confounding effects of tamoxifen. As an alternative, we characterize an adeno associated virus (AAV) with a rat insulin 1 promoter driving Cre recombinase (AAV8 Ins1-Cre) that is economical and rapid to implement, and has limited caveats. Intraperitoneal AAV8 Ins1-Cre produced efficient beta -cell recombination, alongside some hepatic, exocrine pancreas, alpha -cell, delta -cell, and hypothalamic recombination. Delivery of lower doses via the pancreatic duct retained good rates of beta -cell recombination and limited rates of off-target recombination. Unlike inducible Cre in transgenic mice, AAV8 Ins1-Cre required no tamoxifen and premature recombination was avoided. We demonstrate the utility of this technology by inducing hyperglycemia in inducible insulin knockout mice (Ins1(-/-);Ins2(f/f)). AAV-mediated expression of Cre in beta -cells provides an effective alternative to transgenic approaches for inducible knockout studies.