Reid Christopher A, Lipinski Daniel M
Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, WI, USA.
Nuffield Laboratory of Ophthalmology, Department of Clinical Neuroscience, University of Oxford, Oxford, UK.
Methods Mol Biol. 2018;1715:19-31. doi: 10.1007/978-1-4939-7522-8_2.
Over the past two decades recombinant adeno-associated virus (rAAV) vectors have emerged as the gold standard for transferring genetic material to cells of the retina. The ability to effectively produce small batches of rAAV vector at high enough purity for in vitro and in vivo applications in a cost-effective manner is paramount. This is particularly the case when conducting preclinical experiments to screen novel serotypes, promoters or transgenes, where production of numerous vector batches is required. Current vector production methods often produce large quantities of vector, limiting the cost-effectiveness and practicality of such screening experiments, which often require only small volumes of vector to carry out. Herein, we describe a method to produce high titer (10-10 vector genomes (vg)/mL) rAAV vector on small (100 μL) or micro (15 μL) scale for in vitro and in vivo applications.
在过去二十年中，重组腺相关病毒（rAAV）载体已成为将遗传物质转移到视网膜细胞的金标准。以具有成本效益的方式有效生产足够高纯度的小批量rAAV载体以用于体外和体内应用的能力至关重要。在进行临床前实验以筛选新型血清型、启动子或转基因时尤其如此，因为这些实验需要生产大量的载体批次。目前的载体生产方法通常会产生大量载体，限制了此类筛选实验的成本效益和实用性，而这些实验通常只需要少量载体即可进行。在此，我们描述了一种在小（约100μL）或微（约15μL）规模上生产高滴度（10-10载体基因组（vg）/mL）rAAV载体的方法，用于体外和体内应用。
