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Virus Binding and Internalization Assay for Adeno-associated Virus
腺相关病毒的病毒结合和内化测定实验   

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Abstract

The binding and internalization of adeno-associated virus (AAV) is an important determinant of viral infectivity and tropism. The ability to dissect these two tightly connected cellular processes would allow better understanding and provide insight on virus entry and trafficking. In the following protocol, we describe a quantitative PCR (qPCR) based method to determine the amount of vector bound to the cell surface and the amount of subsequent virus internalization based on viral genome quantification. This protocol is optimized for studying AAV. Nevertheless, it can serve as a backbone for studying other viruses with careful modification.

Keywords: Adeno-associated virus(腺相关病毒), Binding(结合), Internalization(内化), Virus entry(病毒进入), Virus(病毒), Cell surface(细胞表面)

Background

Studies that assess AAV biology generally use transgene expression as the experimental endpoint. However, there are a number of critical steps AAV must successfully navigate before it reaches the nucleus and transduces the cell. Therefore, there are multiple distinct steps in the AAV infectious pathway that could be disrupted individually or collectively, leading to altered transduction. Assessment of AAV binding and internalization are important first steps in determining the cause of transduction differences observed upon cellular modification by small molecules, CRISPR-based gene knockout, siRNA-based gene knockdown, or other experimental procedures.

Materials and Reagents

  1. 12-well tissue culture (TC) treated plates (Corning, catalog number: 3513 )
  2. GeneMate 1.7 ml microcentrifuge tubes (BioExpress, catalog number: C-3262-1 )
  3. Tips   
  4. Lightcycler 96-well qPCR plates (Roche Molecular Systems, catalog number: 04729692001 )
  5. Cell lifter (Corning, catalog number: 3008 )
  6. HeLa cells (ATCC, catalog number: CCL-2 )
  7. Purified single-stranded AAV (any serotype) (Grieger et al., 2012)
  8. 1x PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
  9. DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 )
  10. Molecular grade water (Mediatech, catalog number: 46-000-C )
  11. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11995065 )
  12. Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  13. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442 )
  14. 100x penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  15. FastStart Essential DNA Green Master Mix (Roche Molecular Systems, catalog number: 06402712001 )
  16. Virus-specific qPCR primers (at a working concentration of 20 µM each)
    1. fLuc-F – AAAAGCACTCTGATTGACAAATAC
    2. fLuc-R – CCTTCGCTTCAAAAAATGGAAC
  17. Human genomic qPCR primers (at a working concentration of 20 µM each)
    1. hLB2C1-F – GTTAACAGTCAGGCGCATGGGCC
    2. hLB2C1-R – CCATCAGGGTCACCTCTGGTTCC
  18. 10 ng/μl CBA-fLuc plasmid stock solution (see Recipes)
  19. 100 ng/μl HeLa genomic DNA stock solution (see Recipes)

Equipment

  1. Pipette  
  2. Biosafety cabinet
  3. CO2 tissue culture incubator (NuAire, model number: NU-5500 )
  4. Tabletop centrifuge (Eppendorf, catalog number: 022620401 )
  5. Lightcycler 96 qPCR instrument (Roche Molecular Systems, catalog number: 05815916001 )
  6. PCR plate microcentrifuge (VWR, catalog number: 89184-608 )

Procedure

    Notes:
    1. While the use of HeLa cells is described, any cell type can be used, provided the cells remain attached to the plate after several cold PBS washes. If other cell types are used, a proper genomic DNA control will be needed for the qPCR step. Additionally, if non-human cells are used, a set of mLamin primers (or other genomic control) will need to be designed, tested, and used for the qPCR step.
    2. While virus packaging the fLuc transgene is described, any transgene can be used, provided transgene-specific qPCR primers are designed, tested, and used.
    3. Since the binding and internalization assays are separate, the number of wells used must be doubled. Half of the wells will be used for the binding assay, and the other half of the wells will be used for the internalization assay.
    1. Cell preparation for virus binding and internalization assay
      1. Seed 12-well TC plate with 1e5 HeLa cells/well (Figure 1), bringing the well to a final volume of 1 ml. Allow 4-6 h for the cells to fully adhere to the plate at 37 °C in 5% CO2 in a tissue culture incubator.
        Note: If the cells are to be treated with small molecules, treat cells with the compound and vehicle control prior to 4 °C incubation. The amount of incubation time necessary for any small molecule will need to be determined prior to performing this assay.
      2. Incubate cells at 4 °C for 30 min.
      3. Infect cells with AAV at a multiplicity of infection (MOI) of 1e3 vector genomes per cell (vg/cell) by carefully pipetting virus into the meniscus of the media near the edge of the well, taking care not to scrape the well surface. Slowly rock the plate by hand several times to mix.
        Notes:
        1. If the stock concentration of AAV is highly concentrated, the virus can be diluted in DMEM prior to infection.
        2. MOI can be altered to suit individual needs.
      4. Incubate cells at 4 °C for 1 h to allow virus to bind to the cell surface.
      5. Wash cells gently 3 times with 400 µl ice-cold PBS to remove unbound virus by tilting the plate towards you and adding PBS very slowly to the edge of the well with a pipette.
        Note: For each sample, one well will be used for binding, and the other well will be used for internalization.


        Figure 1. Example of experimental plate setup. Example of the plate setup for a binding and internalization experiment. The Trypsin Negative plate is a negative control that is trypsinized immediately after binding is performed.

    2. Binding (1 well)
      1. Add 200 µl PBS to each well.
      2. Scrape off the cells and transfer them to a 1.7 ml microcentrifuge tube.
      3. Isolate total DNA using a QIAGEN DNeasy Blood and Tissue Kit, following manufacturer instructions with the following exception: use 50 µl of molecular grade water to elute sample from the column.
        Note: To increase DNA yield, the water can be heated to 37 °C prior to elution, and the water can be allowed to sit on the membrane for up to 5 min.

    3. Internalization (1 well)
      1. Carefully add 1 ml fresh complete DMEM, pre-warmed to 37 °C, to the cells by tilting the plate towards you and adding very slowly to the edge of the well with a pipette.
      2. Incubate at 37 °C in 5% CO2 in a tissue culture incubator for 1 h to allow for virus internalization.
        Note: The amount of time to allow internalization can be varied, or several time points can be assayed to generate an internalization curve.
      3. Remove the media by aspiration and treat the cells with 1 ml trypsin to detach them from the plate and to remove surface bound virions that did not internalize.
      4. Transfer the trypsinized cells to a 1.7 ml microcentrifuge tube and pellet cells by centrifuging at 500 x g for 3-5 min at room temperature.
      5. Carefully remove trypsin without disturbing the cell pellet.
      6. Wash the cell pellet 3 times by resuspending the cells in 200 µl PBS, pelleting by centrifugation 500 x g for 3-5 min, and carefully removing the PBS.
      7. After the final PBS wash, resuspend the cell pellet in 200 µl PBS.
      8. Isolate total DNA using a QIAGEN DNeasy Blood and Tissue Kit, following manufacturer instructions with the following exception: use 50 µl of molecular grade water to elute sample from the column.

    4. qPCR quantification
      1. fLuc transgene (or transgene of choice):
        1. Generate standard curve for vector genome quantitation using plasmid containing the CBA-fLuc transgene.
          1. Dilute a 10 ng/µl plasmid stock 1:200 in molecular grade water to generate a plasmid solution of 5 x 104 fg/µl.
          2. Perform 7 further serial dilutions of 1:5 in molecular grade water to generate the remainder of the standard curve, with the remaining points of the standard curve having the following concentrations: 1 x 104 fg/µl, 2 x 103 fg/µl, 4 x 102 fg/µl, 8 x 101 fg/µl, 1.6 x 101 fg/µl, 3.2 x 100 fg/µl, and 6.4 x 10-1 fg/µl.
            Note: If there is space on the qPCR plate, 2 standard curves should be used to make quantitation more accurate. In addition, this would allow exclusion of abhorrent points within the standard curve.
        2. Prepare master mix (Table 1) for n + 1 reactions (with n being the total number of reactions).

          Table 1. qPCR master mix

          Note: To prepare a working stock of F + R primers, mix F primer (20 µM concentration) with R primer (20 µM concentration) in a 1:1 ratio. The resulting primer mixture will have both F and R primers at a concentration of 10 µM each.

        3. Load 8 µl master mix in each well.
        4. Load 2 µl of each sample, including standard curve.
          Note: Adding the sample directly to the side of the well assures complete loading of the sample.
        5. Spin the plate in a microplate centrifuge for 30-60 sec at room temperature to assure samples are at the bottom of the wells.
          Note: The VWR microplate centrifuge used has a maximum speed of 500 x g. An adjustable microplate centrifuge can be used at 500-600 x g.
        6. Run the qPCR using the corresponding program in Table 2.

          Table 2. qPCR cycling parameters


      2. hLB2C1 genomic gene
        1. Generate standard curve for cellular genome quantitation using pre-prepared HeLa cell isolated DNA.
          1. Dilute a 100 ng/µl genomic DNA stock solution 1:2 in molecular grade water to generate a genomic DNA solution of 50 ng/µl.
          2. Perform 7 further serial dilutions of 1:2 in molecular grade water to generate the remainder of the standard curve with the remaining points of the standard curve having the following concentrations: 25 ng/µl, 12.5 ng/µl, 6.25 ng/µl, 3.125 ng/µl, 1.563 ng/µl, 0.781 ng/µl, 0.391 ng/µl.
        2. Prepare master mix (Table 1) for n + 1 reactions (with n being the total number of reactions).
        3. Load 8 µl master mix in each well.
        4. Load 2 µl of each sample, including standard curve.
          Note: Adding the sample directly to the side of the well assures complete loading of the sample.
        5. Spin the plate in a microplate centrifuge for 30-60 sec at room temperature to assure samples are at the bottom of the wells.
          Note: The VWR microplate centrifuge used has a maximum speed of 500 x g. An adjustable microplate centrifuge can be used at 500-600 x g.
        6. Run the qPCR using the corresponding program in Table 2.

Data analysis

To calculate the number of vector genomes per cell (Table 3), use the following calculations:

  1. The vector genome copy number is calculated by taking the absolute DNA value derived from the standard curve and multiplying by the number of single-stranded plasmid copies in 1 femtogram of plasmid (270 copies/fg for pTR-CBA-fLuc).
    Note: This value is calculated using the formula below, where 1e15 equals the number of femtograms in a gram, and 650 equals the molecular weight of a DNA base pair. Additionally, the value must be multiplied by a factor of 2 because AAV genomes are single stranded.
    Copy number = 2 x ([6.022 x 1023]/[plasmid size in bp x 1 x 1015 x 650])
  2. The number of diploid genomes is calculated by taking the absolute DNA value derived from the standard curve and dividing by the number of human diploid genomes per nanogram of DNA (167 genomes/ng).
    Note: There is ~6 pg of genomic DNA per diploid human genome. Therefore, in each nanogram of genomic DNA, there are ~167 diploid genomes.
  3. The number of vector genomes per diploid genome is calculated by dividing the total number of vector genomes by the number of diploid genomes.
  4. Binding data is expressed as vg/cell (Figure 2). Internalization can be also expressed as vg/cell. Alternatively, internalization can be expressed as a percentage of bound virions by dividing the number of internalized vg/cell by the number of bound vg/cell (Figure 2).

    Table 3. Sample data analysis

    *Note: This is the absolute DNA value resulting from qPCR with either Luc primers or hLB2C1 primers.

    The P-value is determined using a two-tailed Student’s t-test assuming homoscedasticity. In addition: it is important to run both internalization negative controls (trypsinized cells immediately after binding) and the proper qPCR (water-only) negative controls. Furthermore, independent experiments can be run on the same qPCR plate.


    Figure 2.  Example binding and internalization data from cells treated with the VCP inhibitor Eeyarestatin I (EerI). A. EerI does not alter binding of AAV2 to HeLa cells. B. EerI does not significantly alter internalization of AAV2.

Recipes

  1. 10 ng/μl CBA-fLuc plasmid stock solution
    2 µl plasmid in 398 µl molecular grade water
  2. 100 ng/µl HeLa genomic DNA stock solution (1:2)
    50 µl genomic DNA in 50 µl molecular grade water

Acknowledgments

This study was supported by National Institutes of Health Grants R01HL089221 and P01HL112761 through the NHLBI. This work was also supported by National Institutes of Health Training Grant 5T32 GM007092 through the NIGMS. The protocol described herein was based on the following paper: Berry et al. (2016).

References

  1. Berry, G. E. and Asokan, A. (2016). Chemical modulation of endocytic sorting augments adeno-associated viral transduction. J Biol Chem 291(2): 939-947.
  2. Grieger, J. C. and Samulski, R. J. (2012). Adeno-associated virus vectorology, manufacturing, and clinical applications. Methods Enzymol 507: 229-254.

简介

腺相关病毒(AAV)的结合和内化是病毒感染和向性的重要决定因素。 解决这两个紧密相连的细胞过程的能力将有助于更好地了解并提供有关病毒进入和贩运的洞察。 在以下协议中,我们描述了基于定量PCR(qPCR)的方法,以确定基于病毒基因组定量结合到细胞表面的载体的量和随后的病毒内化的量。 该协议针对研究AAV进行了优化。 然而,它可以作为研究其他病毒,仔细修改的骨干。
【背景】评估AAV生物学的研究通常使用转基因表达作为实验终点。 然而,AAV在到达细胞核并转导细胞之前必须成功导航的许多关键步骤。 因此,AAV感染途径中有多个不同的步骤可能会单独或共同破坏,导致改变的转导。 AAV结合和内化的评估是确定小分子细胞修饰,基于CRISPR的基因敲除,基于siRNA的基因敲除或其他实验程序后观察到的转导差异的原因的重要的第一步

关键字:腺相关病毒, 结合, 内化, 病毒进入, 病毒, 细胞表面

材料和试剂

  1. 12孔组织培养(TC)处理板(Corning,目录号:3513)
  2. GeneMate 1.7ml微量离心管(BioExpress,目录号:C-3262-1)
  3. 提示
  4. Lightcycler 96孔qPCR板(Roche Molecular Systems,目录号:04729692001)
  5. 电池升降机(Corning,目录号:3008)
  6. HeLa细胞(ATCC,目录号:CCL-2)
  7. 纯化的单链AAV(任何血清型)(Grieger等人,2012)
  8. 1x PBS(Thermo Fisher Scientific,Gibco TM,目录号:14190144)
  9. DNeasy血液和组织试剂盒(QIAGEN,目录号:69504)
  10. 分子级水(Mediatech,目录号:46-000-C)
  11. DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:11995065)
  12. 胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25300054)
  13. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F2442)
  14. 100x青霉素/链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  15. FastStart Essential DNA Green Master Mix(Roche Molecular Systems,目录号:06402712001)
  16. 病毒特异性qPCR引物(每个工作浓度为20μM)
    1. fLuc-F - AAAAGCACTCTGATTGACAAATAC
    2. fLuc-R - CCTTCGCTTCAAAAAATGGAAC
  17. 人基因组qPCR引物(每个工作浓度为20μM)
    1. hLB2C1-F-GTTAACAGTCAGGCGCATGGGCC
    2. hLB2C1-R - CCATCAGGGTCACCTCTGGTTCC
  18. 10 ng /μlCBA-fLuc质粒原液(见配方)
  19. 100 ng /μlHeLa基因组DNA储备溶液(见配方)

设备

  1. 移液器
  2. 生物安全柜
  3. CO 2组织培养培养箱(NuAire,型号:NU-5500)
  4. 台式离心机(Eppendorf,目录号:022620401)
  5. Lightcycler 96 qPCR仪器(Roche Molecular Systems,目录号:05815916001)
  6. PCR板微量离心机(VWR,目录号:89184-608)

程序

    注意:
    1. 虽然描述了使用HeLa细胞,但是可以使用任何细胞类型,只要细胞在几次冷PBS洗涤后保持连接到平板上即可。如果使用其他细胞类型,则qPCR步骤将需要适当的基因组DNA控制。另外,如果使用非人细胞,则需要设计,测试和使用一组mLamin引物(或其他基因组对照)并用于qPCR步骤。
    2. 描述了包装fLuc转基因的病毒包装,只要设计,测试和使用转基因特异性qPCR引物,就可以使用任何转基因。
    3. 由于结合和内化测定是分开的,因此使用的孔数必须加倍。一半的孔将用于结合测定,另一半的孔将用于内化测定。
    1. 用于病毒结合和内化测定的细胞制备
      1. 种子12孔TC板,1e5 HeLa细胞/孔(图1),使孔达到1毫升的最终体积。允许4-6小时使细胞在37℃下在组织培养箱中的5%CO 2中完全粘附到板上。
        注意:如果要用小分子处理细胞,在4℃孵育之前用化合物和载体对照处理细胞。在进行此测定之前,需要确定任何小分子所需的孵育时间。
      2. 在4℃孵育细胞30分钟
      3. 通过仔细将病毒移入孔边缘附近的培养液的半月板,以感染复数(MOI)1e3载体基因组每个细胞(vg /细胞)感染细胞,注意不要刮掉孔表面。用手慢慢摇动板块混合。
        注意:
        1. 如果AAV的库存浓度高度浓缩,则病毒可以在感染前在DMEM中稀释。
        2. 可以修改MOI以适应个人需要。
      4. 在4℃下孵育细胞1小时,以使病毒与细胞表面结合
      5. 用400μl冰冷的PBS轻轻洗涤细胞3次以通过将板倾斜向上移动未移除的病毒,并用移液管将PBS缓慢加入到孔的边缘。
        注意:对于每个样本,一个井将用于绑定,另一个井将用于内部化。


        图1.实验板设置示例。用于结合和内化实验的板设置示例。胰蛋白酶阴性板是在结合后立即进行胰蛋白酶消化的阴性对照。

    2. 绑定(1孔)
      1. 每孔加入200μlPBS。
      2. 刮掉细胞并将其转移到1.7 ml的微量离心管中
      3. 使用QIAGEN DNeasy血液和组织试剂盒分离总DNA,遵循制造商的说明书,但有以下例外:使用50μl分子级水从柱中洗脱样品。
        注意:为了提高DNA产量,可以在洗脱前将水加热至37℃,并将水置于膜上长达5分钟。

    3. 内化(1井)
      1. 小心地将1ml新鲜的完全DMEM预热至37℃,通过将板倾斜向细胞,并用移液管非常缓慢地加入到孔的边缘。
      2. 在组织培养箱中于37℃在5%CO 2中孵育1小时以允许病毒内化。
        注意:允许内部化的时间可以改变,或者可以测定几个时间点来产生内部曲线。
      3. 通过抽吸取出培养基,并用1ml胰蛋白酶处理细胞,将其从板上分离出来,并除去未内化的表面结合的病毒粒子。
      4. 将胰蛋白酶处理的细胞转移到1.7ml微量离心管中,并通过在室温下以500×g离心3-5分钟来沉淀细胞。
      5. 仔细清除胰蛋白酶,不会干扰细胞沉淀
      6. 通过将细胞重新悬浮在200μlPBS中3次洗涤细胞沉淀,通过离心500×g离心3-5分钟,并小心地除去PBS。
      7. 最后PBS洗涤后,将细胞沉淀重悬于200μlPBS中
      8. 使用QIAGEN DNeasy血液和组织试剂盒分离总DNA,遵循制造商的说明书,但有以下例外:使用50μl分子级水从柱中洗脱样品。

    4. qPCR定量
      1. 转基因(或选择的转基因):
        1. 使用含有CBA-fLuc转基因的质粒产生载体基因组定量的标准曲线。
          1. 在分子级水中稀释10ng /μl质粒原液1:200,以产生5×10 4个/μg/μl的质粒溶液。
          2. 在分子级水中进行7次进一步的1:5的系列稀释,以产生标准曲线的其余部分,其中标准曲线的剩余点具有以下浓度:1×10 4/ff /μl, 2×10 3 /分钟fg /μl,4×10 2/ff /μl,8×10 fg /μl,1.6×10 1 fg /μl,3.2×10 0 fg /μl和6.4×10 fg /μl。
            注意:如果qPCR板上有空格,应使用2个标准曲线使定量更准确。此外,这将允许在标准曲线内排除令人讨厌的点。
        2. 为n + 1反应准备主混合物(表1)(n为反应总数)。

          表1. qPCR主混合

          注意:为了制备F + R引物的工作原料,将F引物(20μM浓度)与R引物(20μM浓度)以1:1的比例混合。所得到的引物混合物将具有浓度为10μM的F和R引物。

        3. 在每个孔中加载8μl主混合物。
        4. 加载2μl每个样品,包括标准曲线。
          注意:将样品直接添加到孔的一侧可确保样品的完整载入。
        5. 在平板离心机中将板旋转30-60秒,以确保样品位于孔底部。
          注意:使用的VWR微孔板离心机的最大速度为500 x g。可调式微板离心机可用于500-600 x g。
        6. 使用表2中的相应程序运行qPCR。

          表2. qPCR循环参数


      2. hLB2C1基因组基因
        1. 使用预先制备的HeLa细胞分离的DNA产生细胞基因组定量的标准曲线。
          1. 在分子级水中稀释100ng /μl基因组DNA储备液1:2,以产生50 ng /μl的基因组DNA溶液。
          2. 在分子级水中进一步进行1:2的连续稀释,产生标准曲线的剩余部分,标准曲线的其余部分具有以下浓度:25ng /μl,12.5ng /μl,6.25ng /μl,3.125 ng /μl,1.563ng /μl,0.781ng /μl,0.391ng /μl
        2. 为n + 1反应准备主混合物(表1)(n为反应总数)。
        3. 在每个孔中加载8μl主混合物。
        4. 加载2μl每个样品,包括标准曲线。
          注意:将样品直接添加到孔的一侧可确保样品的完整载入。
        5. 在平板离心机中将板旋转30-60秒,以确保样品位于孔底部。
          注意:使用的VWR微孔板离心机的最大速度为500 x g。可调式微板离心机可用于500-600 x g。
        6. 使用表2中的相应程序运行qPCR。

数据分析

要计算每个细胞的载体基因组数(表3),请使用以下计算:

  1. 通过取从标准曲线得到的绝对DNA值乘以1个质粒(270拷贝/fg,pTR-CBA-fLuc)中的单链质粒拷贝的数目来计算载体基因组拷贝数。 > 注意:该值使用以下公式计算,其中1e15等于一克中的飞镖数,650等于DNA碱基对的分子量。另外,由于AAV基因组是单链的,所以该值必须乘以2因子。
    拷贝数= 2×([6.022×10 23 ]/[质粒大小(bp×1×10 15)×650])
  2. 通过从标准曲线得到的绝对DNA值除以每毫克DNA(167个基因组/ng)的人类二倍体基因组数目来计算二倍体基因组数目。
    注意:每个二倍体人类基因组有约6 pg的基因组DNA。因此,在每个纳克基因组DNA中,有〜167个二倍体基因组。
  3. 通过将载体基因组的总数除以二倍体基因组的数目来计算每个二倍体基因组的载体基因组数量。
  4. 绑定数据表示为vg/cell(图2)。内部化也可以表示为vg/cell。或者,通过将内化的vg /细胞数除以结合的vg /细胞的数目,内化可以表示为结合的病毒粒子的百分比(图2)。

    表3.样本数据分析

    注意:这是使用Luc引物或hLB2C1引物的qPCR产生的绝对DNA值。

    使用双尾学生的假设均衡性来确定 P - 值。另外:重要的是运行内化阴性对照(结合后立即胰蛋白酶化细胞)和正确的qPCR(纯水)阴性对照。此外,独立实验可以在相同的qPCR板上运行。


    图2.使用VCP抑制剂Eeyarestatin I(EerI)处理的细胞的结合和内化数据的实例.EerI不改变AAV2与HeLa细胞的结合。 B.EerI不会显着改变AAV2的内在化。

食谱

  1. 10 ng /μlCBA-fLuc质粒原液
    在398μl分子级水中的2μl质粒
  2. 100ng /μlHeLa基因组DNA储备液(1:2)
    50μl分子级水中的50μl基因组DNA

致谢

本研究由国家卫生研究院R01HL089221和P01HL112761通过NHLBI支持。这项工作也得到了国家卫生研究院培训基金会通过NIGMS 5T32 GM007092的支持。本文描述的方案基于以下论文:Berry等人(2016)。

参考文献

  1. Berry,GE和Asokan,A.(2016)。 内吞分选的化学调节增强腺相关病毒转导。 J Biol Chem 291(2):939-947。
  2. Grieger,JC和Samulski,RJ(2012)。  Adeno 相关的病毒载体学,制造和临床应用。 方法Enzymol 507:229-254。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Berry, G. E. and Tse, L. V. (2017). Virus Binding and Internalization Assay for Adeno-associated Virus. Bio-protocol 7(2): e2110. DOI: 10.21769/BioProtoc.2110.
  2. Berry, G. E. and Asokan, A. (2016). Chemical modulation of endocytic sorting augments adeno-associated viral transduction. J Biol Chem 291(2): 939-947.
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