Adeno-associated viral serotypes produce differing titers and differentially transduce neurons within the rat basal and lateral amygdala
Adeno-associated viral serotypes produce differing titers and differentially transduce neurons within the rat basal and lateral amygdala
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DOI：
10.1186/1471-2202-15-28
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发表时间：
2014-02-18
期刊：
影响因子：
2.4
通讯作者：
Ploski, Jonathan E.
中科院分区：
文献类型：
--
作者：
Holehonnur, Roopashri;Luong, Jonathan A.;Ploski, Jonathan E.
背景资料：近年来，利用重组腺相关病毒（AAV）在啮齿动物脑内进行局部遗传操作的兴趣越来越大。AAV的不同血清型具有不同的衣壳蛋白序列，并且这些变化极大地影响每种血清型对特定细胞类型和脑区域的免疫能力。因此，我们的目的是确定对于靶向基底和外侧杏仁核（BLA）内的神经元最佳的AAV血清型，因为先前尚未在BLA内检查AAV的转导效率。由于该区域在情感、学习和记忆以及许多精神疾病中的作用，因此希望对其进行遗传操纵。我们通过筛选9种不同的AAV血清型来实现这一点AAV 2/1、AAV 2/2、AAV 2/5、AAV 2/7、AAV 2/8、AAV 2/9、AAV 2/rh 10、AAV 2/DJ和AAV 2/DJ 8），其被设计成在α Ca 2 +/钙调蛋白依赖性蛋白激酶II启动子（aCaMKII）的调控下表达红色荧光蛋白（RFP）。我们确定这些血清型在标准实验室生产下产生不同量的病毒。值得注意的是，与检查的其他血清型相比，AAV 2/2始终产生最低滴度。将这9种血清型以每种血清型达到的最高滴度和7.8E + 11 GC/ml的标准化滴度双侧输注到大鼠BLA中。病毒输注后21天，在整个杏仁核中定量转导的程度。这些病毒在BLA内表现出不同的神经元转导。与检查的血清型相比，AAV 2/7表现出具有最高转导效率的趋势，而AAV 2/5表现出显著较低的转导效率。AAV 2/5的能力下降，以抑制BLA神经元与其显着不同的衣壳蛋白序列相比，其他血清型examined.Conclusions：对于实验室生产自己的重组腺相关病毒，使用AAV 2/2可能是不太可取的，因为AAV 2/2产生显着较低的滴度比许多其他血清型的AAV。除了AAV 2/5之外，许多AAV血清型似乎有效地刺激BLA神经元。考虑到某些血清型实现高滴度和良好的BLA神经元的能力，在我们手中，AAV 2/DJ 8和AAV 2/9似乎是当靶向BLA内的神经元时使用的理想血清型。
Background: In recent years, there has been an increased interest in using recombinant adeno-associated viruses (AAV) to make localized genetic manipulations within the rodent brain. Differing serotypes of AAV possess divergent capsid protein sequences and these variations greatly influence each serotype's ability to transduce particular cell types and brain regions. We therefore aimed to determine the AAV serotype that is optimal for targeting neurons within the Basal and Lateral Amygdala (BLA) since the transduction efficiency of AAV has not been previously examined within the BLA. This region is desirable to genetically manipulate due to its role in emotion, learning & memory, and numerous psychiatric disorders. We accomplished this by screening 9 different AAV serotypes (AAV2/1, AAV2/2, AAV2/5, AAV2/7, AAV2/8, AAV2/9, AAV2/rh10, AAV2/DJ and AAV2/DJ8) designed to express red fluorescent protein (RFP) under the regulation of an alpha Ca2+/calmodulin-dependent protein kinase II promoter (aCaMKII).Results: We determined that these serotypes produce differing amounts of virus under standard laboratory production. Notably AAV2/2 consistently produced the lowest titers compared to the other serotypes examined. These nine serotypes were bilaterally infused into the rat BLA at the highest titers achieved for each serotype and at a normalized titer of 7.8E + 11 GC/ml. Twenty one days following viral infusion the degree of transduction was quantitated throughout the amygdala. These viruses exhibited differential transduction of neurons within the BLA. AAV2/7 exhibited a trend toward having the highest efficiency of transduction and AAV2/5 exhibited significantly lower transduction efficiency as compared to the serotypes examined. AAV2/5's decreased ability to transduce BLA neurons correlates with its significantly different capsid protein sequences as compared to the other serotypes examined.Conclusions: For laboratories producing their own recombinant adeno-associated viruses, the use of AAV2/2 is likely less desirable since AAV2/2 produces significantly lower titers than many other serotypes of AAV. Numerous AAV serotypes appear to efficiently transduce BLA neurons, with the exception of AAV2/5. Taking into consideration the ability of certain serotypes to achieve high titers and transduce BLA neurons well, in our hands AAV2/DJ8 and AAV2/9 appear to be ideal serotypes to use when targeting neurons within the BLA.