AAV8, 9, Rh10, Rh43 vector gene transfer in the rat brain: Effects of serotype, promoter and purification method
AAV8, 9, Rh10, Rh43 vector gene transfer in the rat brain: Effects of serotype, promoter and purification method
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DOI：
10.1038/sj.mt.6300331
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发表时间：
2008-01-01
影响因子：
12.4
通讯作者：
Henning, Phillip P.
中科院分区：
文献类型：
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作者：
Klein, Ronald L.;Dayton, Robert D.;Henning, Phillip P.
我们比较了腺相关病毒(AAV)血清型绿色荧光蛋白(GFP)在成年大鼠海马区的表达水平。用CSCL法纯化含有巨细胞病毒启动子驱动的GFP的AAV血清型8、9、RH10和Rh43。以AAV8为参照，AAVRH10和AAVRh43产生的GFP水平都不高于AAV8。对于AAV9，与AAV8相比有所增加。CSCL纯化的AAV8呈现星形胶质细胞转导模式，与其他AAVs预期的神经元表达相反。在AAV8中用碘二醇纯化法制备了相同的CMV-GFP质粒后，得到了预期的神经元模式。CSCL AAV8星形胶质细胞的表达可能是由于相对较高的蛋白质杂质水平所致。我们比较了CMV启动子和CMV/鸡β-肌动蛋白(CBA)启动子在AAV8中的表达情况，发现CBA启动子能够产生更强的GFP表达。在对AAV8、AAV9和AAVRH10进行血清型、启动子和纯化优化的两种剂量的载体上，我们没有观察到AAV8、AAV9和AAVRH10的血清型差异。因此，当比较血清型强度时，纯化方法会影响转导模式和结果。
We compared adeno-associated virus (AAV) serotypes for expression levels of green fluorescent protein (GFP) in the adult rat hippocampus by biophotonic imaging. Preparations of AAV serotypes 8, 9, Rh10, and Rh43 incorporating cytomegalovirus (CMV) promoter-driven GFP were purified by a CsCl method. Neither AAV Rh10 nor AAV Rh43 produced greater levels of GFP than AAV8, which was used as a reference. For AAV9, there was an increase relative to AAV8. The CsCl-purified AAV8 displayed an astroglial transduction pattern in contrast to the expected neuronal expression of other AAVs. After preparing the same CMV-GFP plasmid in AAV8 with an iodixanol purification method, the expected neuronal pattern resulted. The astroglial expression with the CsCl AAV8 was probably due to relatively high levels of protein impurities. We compared the CMV promoter with the CMV/chicken beta-actin (CBA) promoter in the context of AAV8, both prepared by iodixanol, and found the CBA promoter to produce stronger GFP expression. At two doses of vectors optimized for serotype, promoter and purification, we did not observe serotype differences among AAV8, AAV9, or AAV Rh10. The purification method can therefore impact the transduction pattern as well as the results when comparing serotype strengths.