Abstract
In vivo genetic manipulation is used to study the impact of gene deletion or re-expression on β-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 β-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 β-cell recombination, alongside some hepatic, exocrine pancreas, α-cell, δ-cell, and hypothalamic recombination. Delivery of lower doses via the pancreatic duct retained good rates of β-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−/−;Ins2f/f). AAV-mediated expression of Cre in β-cells provides an effective alternative to transgenic approaches for inducible knockout studies.
Highlights
In vivo genetic manipulation is used to study the impact of gene deletion or re-expression on β-cell function and organism physiology
As several reports have already shown that transgenic insulin 2 (Ins2)-Cre mice may develop glucose intolerance[4], we studied glucose metabolism over 8 weeks (−1 to 7 weeks relative to associated virus (AAV) administration)
We propose that the AAV could be used as a vector for delivery of Cre to β-cells
Summary
In vivo genetic manipulation is used to study the impact of gene deletion or re-expression on β-cell function and organism physiology. Alternative lines make use of a pancreatic and duodenal homeobox 1 gene (Pdx1) promoter to control Cre expression, but it has been reported to induce hypothalamic[7], α-cell, and δ-cell recombination[8] To address this issue, a more specific Ins[1] promoter has been used and may limit these off-target confounding effects[9,10]. Finding a way to avoid caveats of developmental deletion of LoxP flanked genes, GH minigene-induced β-cell dysfunction, unintended recombination before tamoxifen administration, and tamoxifen toxicity would be useful. It is worth noting the time and costs of crossing Cre driver mice with LoxP containing mice to generate mice with suitable genotypes. AAV delivered via the pancreatic duct at ~1/10th the dose of IP delivery can infect β-cells at comparable efficiency[23]
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