Kemter Elisabeth, Citro Antonio, Wolf-van Buerck Lelia, Qiu Yi, Böttcher Anika, Policardi Martina, Pellegrini Silvia, Valla Libera, Alunni-Fabbroni Marianna, Kobolák Julianna, Kessler Barbara, Kurome Mayuko, Zakhartchenko Valeri, Dinnyes Andras, Cyran Clemens C, Lickert Heiko, Piemonti Lorenzo, Seissler Jochen, Wolf Eckhard
Department of Veterinary Sciences and Gene Center, Chair for Molecular Animal Breeding and Biotechnology, LMU Munich, Munich, Germany.
Department of Veterinary Sciences, Center for Innovative Medical Models (CiMM), LMU Munich, Oberschleißheim, Germany.
Xenotransplantation. 2022 Jan;29(1):e12719. doi: 10.1111/xen.12719. Epub 2021 Dec 21.
Islet xenotransplantation is a promising concept for beta-cell replacement therapy. Reporter genes for noninvasive monitoring of islet engraftment, graft mass changes, long-term survival, and graft failure support the optimization of transplantation strategies. Near-infrared fluorescent protein (iRFP) is ideal for fluorescence imaging (FI) in tissue, but also for multispectral optoacoustic tomography (MSOT) with an even higher imaging depth. Therefore, we generated reporter pigs ubiquitously expressing iRFP.
CAG-iRPF720 transgenic reporter pigs were generated by somatic cell nuclear transfer from FACS-selected stable transfected donor cells. Neonatal pig islets (NPIs) were transplanted into streptozotocin-diabetic immunodeficient NOD-scid IL2Rg (NSG) mice. FI and MSOT were performed to visualize different numbers of NPIs and to evaluate associations between signal intensity and glycemia. MSOT was also tested in a large animal model.
CAG-iRFP transgenic NPIs were functionally equivalent with wild-type NPIs. Four weeks after transplantation under the kidney capsule, FI revealed a twofold higher signal for 4000-NPI compared to 1000-NPI grafts. Ten weeks after transplantation, the fluorescence intensity of the 4000-NPI graft was inversely correlated with glycemia. After intramuscular transplantation into diabetic NSG mice, MSOT revealed clear dose-dependent signals for grafts of 750, 1500, and 3000 NPIs. Dose-dependent MSOT signals were also revealed in a pig model, with stronger signals after subcutaneous (depth ∼6 mm) than after submuscular (depth ∼15 mm) placement of the NPIs.
Islets from CAG-iRFP transgenic pigs are fully functional and accessible to long-term monitoring by state-of-the-art imaging modalities. The novel reporter pigs will support the development and preclinical testing of novel matrices and engraftment strategies for porcine xeno-islets.
胰岛异种移植是一种很有前景的β细胞替代治疗概念。用于无创监测胰岛植入、移植物质量变化、长期存活和移植物失败的报告基因有助于优化移植策略。近红外荧光蛋白（iRFP）不仅适用于组织中的荧光成像（FI），还适用于成像深度更高的多光谱光声断层扫描（MSOT）。因此，我们培育出了普遍表达iRFP的报告猪。
通过体细胞核移植技术，从经荧光激活细胞分选术（FACS）筛选出的稳定转染供体细胞中培育出CAG-iRPF720转基因报告猪。将新生猪胰岛（NPI）移植到链脲佐菌素诱导糖尿病的免疫缺陷NOD-scid IL2Rg（NSG）小鼠体内。进行FI和MSOT以可视化不同数量的NPI，并评估信号强度与血糖之间的关联。MSOT也在大型动物模型中进行了测试。
CAG-iRFP转基因NPI在功能上与野生型NPI相当。肾包膜下移植四周后，FI显示4000个NPI的移植物信号比1000个NPI的移植物高两倍。移植十周后，4000个NPI移植物的荧光强度与血糖呈负相关。将NPI肌肉内移植到糖尿病NSG小鼠体内后，MSOT显示750、1500和3000个NPI移植物有明显的剂量依赖性信号。在猪模型中也显示出剂量依赖性MSOT信号，皮下（深度约6毫米）植入NPI后的信号比肌肉下（深度约15毫米）植入后的信号更强。
CAG-iRFP转基因猪的胰岛功能完全正常，可通过先进的成像方式进行长期监测。这种新型报告猪将有助于开发和临床前测试用于猪异种胰岛的新型基质和植入策略。
