Bac-N-Blue™ Transfection Kit - FAQs

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106 product FAQs found

白色菌落不能在液体培养基中生长。我该怎么办？

庆大霉素浓度可能过高。尝试降低庆大霉素的浓度至5 µg/mL，并在培养基中加入更多菌落。

为什么会出现蓝色菌落？为什么会出现中心为蓝色而边缘为白色的菌落？

若出现蓝色菌落，则大肠杆菌含有杆粒和质粒，使细胞能在筛选过程中存活。但是，由于未发生转座，所以LacZ基因未被破坏。靶心菌落表示在菌落生长时发生转座。将从混合克隆白色部分中分离得到的克隆重新划线，应该能够得到发生过转座的菌落。

我得到的主要是白色/野生型空斑，而不是蓝色/重组型空斑。哪里出错了？

这是同源性重组较差的典型标志。应检查使用的质粒/线性DNA比例。但是，如果存在一些蓝色空斑，则对那些病毒进行扩增并检测它们的蛋白。根据我们的经验，它们应该是正确的，即使其丰度相对较低。

感染细胞后，我在一个细胞中看到较大的多角体，而在相邻细胞中看到较小的多角体（数量更多）。这正常吗？

是的，细胞被单个野生型病毒感染后，会以不同速度生成多角体，直到培养瓶中所有的细胞都被感染。细胞中多角体的形成需要3-4天左右，其大小和数量各不相同，直至达到最大能力并发生细胞破裂，从而将微小的病毒颗粒释放到培养基中。

我担心得不到空斑。出现空斑需要几天？空斑通常为多大？

正常情况下，约5-7天会出现很小的白点，约10天出现1 mm空斑。空斑的大小范围为1 mm至4 mm。

我忘记在培养板中加入Bluo-gal了。我能否稍后再加？

在打算挑选空斑的那一天，制备含Bluo-gal的DMSO溶液，浓度为20 mg/mL。在每个板中加入50 µL，使用玻璃涂布器在无菌条件下涂布。等待30-60分钟，空斑应变为蓝色。

什么原因会导致培养板变蓝并形成蓝色空斑？

有几种原因可导致培养板变蓝：

•病毒接种量过多。可尝试使用更高的稀释度。
•加入热的融化琼脂糖时，将细胞烫死了。过热的琼脂糖会使细胞裂解，将lacZ释放到琼脂糖中，使其变蓝。应复查铺板温度。如果培养板过湿，蓝色会扩散。

在细胞上覆盖琼脂糖后，细胞呈新月形或颗粒状。这是为什么？

琼脂糖覆层过热。加入琼脂糖覆层后，细胞应仍然保持圆形和健康状态。

我的琼脂糖覆层漂起来了。这是因为我没有将培养基完全吸走吗？

是的，这表明空斑上存在吸液问题。琼脂糖覆层“漂浮”，是因为板中的培养基未被全部吸走。将琼脂糖覆盖到细胞上之前，培养板应完全干燥，特别是在将要挑选空斑时。为达到这样的效果，我们通常会将培养板稍微倾斜，用Pasteur移液管沿培养板边缘吸一圈，同时小心不要破坏细胞单层。如果培养板边缘有任何培养基残留（小液滴），则继续吸液。琼脂糖覆层“漂浮”，也可能导致野生型病毒污染。野生型病毒可以迁移到培养板的其他部分，从而污染重组型空斑。野生型病毒比重组型病毒复制得快的多，可迅速超过重组型病毒。

我看到了类似于小空斑的东西，但很难看清楚。问题出在哪里？

细胞接种过多；我们建议在6孔板的每孔中接种8 x 105个细胞。

我觉得我加入了过多的病毒。MOI有多重要？病毒用量过多会导致什么？

通常使用的MOI在5-10之间。如果加入过多病毒，会导致细胞过早死亡，并使蛋白表达水平降低。

我的杆状病毒空斑实验未得到空斑。可能原因是什么？

感染动力学可能比预期要慢。继续观察培养板，直至感染后第8-9天。如果未出现空斑，则检查下面各项：

•如果细胞不健康，会产生较差的空斑或无空斑产生。理想状态下，细胞应处于对数生长中期，并且存活率大于90%。在感染导致细胞停止生长前，细胞应至少倍增1次。应确保细胞接种量正确，且汇合度约为70%。
•细胞营养不良和健康状态较差，都会抑制病毒复制周期。
•琼脂糖温度也很关键。在琼脂糖覆层后，应将培养板静置1小时，使琼脂糖完全凝固。
•在27°C孵育时，过度冷凝会抑制空斑形成——一旦发生冷凝，就应移除纸巾或打开装有培养板的容器。
•病毒滴度过低：使用较高的病毒滴度。您可能需要再次感染细胞，并收集更高滴度的病毒储液。

我在使用Bac-N-Blue表达系统后，对来自多个空斑的重组病毒进行PCR分析，结果发现存在野生型和重组型病毒的混合物。我该怎么办？

如果您所有的样品中都有野生型病毒污染，则使用P1病毒储液重新进行空斑实验。应确保选择间隔合适的、occ-空斑。如果您难以对重组空斑和非重组空斑进行区分，可尝试使用一种pBlueBac载体。在琼脂糖覆层时，若在培养基中加入显色底物，则重组空斑为蓝色（想要获取更多信息，请参见使用手册第14页）。

使用Bac-N-Blue表达系统时，在感染后4天依然未见到病毒感染迹象。我该怎么办？

请对比仅有细胞的培养板和感染的培养板。与感染的细胞相比，未感染的细胞应表现为过度生长状态，因为转染会抑制生长。如果出现这种情况，应继续每日观察感染细胞是否有其他感染迹象（核肿胀、脱离培养板、病毒出芽以及裂解）。感染动力学可能比预期慢。如果细胞生长未出现抑制现象，您可考虑以下因素：

•DNA是如何提取的？我们建议使用树脂DNA纯化系统，例如我们的PureLink HiPure质粒提取试剂盒。
•细胞是否处于对数期？汇合度是多少？为实现成功转染，我们建议使用处于对数生长期的细胞，存活率至少为95%，并以50-70%的汇合度进行接种。
•使用的转染试剂是什么？我们建议使用Cellfectin II试剂。

使用BaculoDirect表达试剂盒后，无法检测到任何重组融合蛋白。可能原因是什么？你们有何建议？

请检验入门克隆的构建，应确保插入片段位于载体读码框内。在LR反应后，利用PCR法分析重组病毒DNA，确认插入片段的大小和方向均正确。对PCR产物进行测序，确认用于表位标签表达的读码框正确。

我的P1病毒储液滴度较低，如何能够生成高滴度储液？

为得到高滴度储液，应使用P1储液再次感染细胞并生成P2高滴度储液。按照BaculoDirect使用手册第18页的说明，生成P2储液。

我进行了LR反应，并随后转染到Sf9/Sf21昆虫细胞中，但即使72小时后仍未观察到任何感染迹象。我该怎么办？

请查看以下建议：

•在转染到昆虫细胞中之前，利用PCR分析检验LR反应。
•在转染实验中，我们建议使用无添加的Grace’s昆虫细胞培养基代替无血清培养基，因为无血清培养基中的有些成分可能干扰转染。
•转染期间，应保证无FBS、无添加剂、无抗生素，因为这些物质中的蛋白质会干扰Cellfectin II试剂。
•LR重组反应中，以pENTR/CAT质粒为阳性对照，以Cellfectin II试剂（模拟转染）作为阴性对照。
•确保细胞处于对数生长期，存活率高于95%，细胞使用量应与使用手册的建议一致。
•若转染效率较低，细胞可能需要1周时间才能出现病毒感染迹象；继续培养，同时每日观察细胞的感染迹象。

更昔洛韦溶液中出现沉淀。我该怎么办？

在37°C水浴中温浴更昔洛韦5-10分钟，然后涡旋数分钟。沉淀应该重新溶解于溶液中。

我在使用His标签纯化柱对在昆虫细胞中表达的分泌型蛋白进行纯化时，未得到蛋白。为什么？我该怎么办？

用于培养昆虫细胞的培养基通常具有酸性pH（6.0-6.5）或含有电子供应基团，可阻止6xHis标签蛋白与Ni-NTA发生结合。昆虫细胞生长培养基所含谷氨酰胺、甘氨酸或组氨酸等氨基酸的浓度显著高于哺乳细胞生长培养基，这些氨基酸可与6xHis标签蛋白竞争Ni-NTA基质上的结合位点。例如，Grace’s培养基（Life Technologies）含有约10 mM谷氨酰胺、10 mM甘氨酸和15 mM组氨酸。

使用含适当成分和pH（8.0）的缓冲液对培养基进行透析，透析用缓冲液与在天然条件下进行纯化所使用的推荐裂解缓冲液相似，通常可恢复最佳的结合条件。应注意，选用的培养基可能导致出现白色沉淀（可能由不溶性盐组成），但6xHis标签蛋白通常留在溶液中。使用无蛋白质的培养基时，可通过蛋白质定量进行检测；或者利用免疫印迹分析法检测6xHis标签蛋白的含量。离心后，可从清澈的上清液中直接纯化6xHis标签蛋白。

从Bac-to-Bac杆状病毒表达系统得到的病毒储液一开始效果很好，但是几个月之后，蛋白表达明显降低。这是为什么？

请查看以下可能原因和解决方案：

•使用较高的起始MOI扩增病毒储液：使用低代数病毒储液，进行低MOI扩增。
•收获病毒上清液时，未离心和除去细胞：重新使用低代数病毒储液，进行低MOI扩增；如果该病毒储液代数为P2，则该储液可用于扩增。
•某些基因可使病毒变得非常不稳定：拿出分装的P2病毒储液，复苏后进行扩增。

使用Bac-to-Bac杆状病毒表达系统时，蛋白得率较低。可能原因是什么？

蛋白得率较低可能是因为：

•病毒储液含有重组和非重组杆状病毒的混合物：通过空斑纯化，分离重组杆状病毒。
•杆状病毒不是重组的：利用pUC/M13正向和反向引物对杆粒DNA进行PCR分析，对转座进行验证；使用新的重组杆粒DNA再次转染昆虫细胞。
•病毒滴度过低或过高：改变MOI。
•收获细胞的时间点不理想：采用针对表达的时间进程实验，确定获得最高蛋白表达水平的最佳时间。
•细胞生长条件和培养基不理想：根据培养瓶的尺寸和表达条件，优化细胞培养条件；为获得最佳的细胞生长和蛋白表达，我们建议使用Sf-900 II SFM或Sf-900 III SFM。
•所用细胞系不理想；尝试使用其他昆虫细胞系。
•细胞收获过晚：做一个时间进程实验，在不同时间点收获细胞。

我使用P2病毒储液感染细胞72小时后，未看到任何细胞感染迹象。这是为什么？

检查MOI。P1病毒的滴度低于估计值，可能导致MOI较低。

使用Bac-to-Bac杆状病毒表达系统，转染细胞后未看到任何病毒产生的迹象。已经5天了。这是为什么？该怎么解决？

请查看以下可能原因和建议：

•Cellfectin II试剂与杆粒没有混合或孵育时间不足：通过敲打或轻轻涡旋，混匀Cellfectin II试剂与杆粒，孵育混合物15-45分钟。
•杆粒得率低于预期：使用不同剂量的杆粒进行优化。
•杆粒在纯化或冻融过程中被剪切：在凝胶上验证杆粒的完整性。
•孵育时间不足：将混合物在27°C孵育8小时。
•所用细胞代数过高或已过对数生长期：为获得最佳结果，使用代数在8-15代之间的细胞；接种时，细胞应处于对数生长期。
•Cellfectin II试剂被冷冻过：购买新的试剂。
•所用培养基含有血清：转染时，使用无添加的Grace’s培养基。

已转染的细胞和只加了Cellfectin II试剂的阴性对照组细胞都在36小时内发生死亡。可能的原因是什么？

有多种可能原因：

•转染所用培养基含有抗生素。
•细胞接种密度过低：我们建议细胞汇合度至少达到70%。
•所用细胞代数过低：我们建议细胞用于转染前，至少生长5代。
•转染后未使用青霉素/链霉素，导致污染：加入转染混合物孵育5-8小时后，移除混合物，并在每孔中加入含有抗生素的培养基。

转染后细胞漂浮，看起来已死亡，但只加了Cellfectin II试剂的对照板是正常的。这是为什么？

这可能是因为来自杆粒制备的污染或细胞毒性。应设置无Cellfectin II试剂的杆粒阴性对照组。此外，在制备杆粒时，应使用PureLink HiPure质粒制备试剂盒，而不是基于硅胶的小量提取试剂盒。

旧的低滴度储液能否用于制备高滴度储液？

如果该低滴度储液为P1或P2储液，则可用于病毒扩增实验方案。如果低滴度储液曾经是高滴度储液，但随着时间或繁殖多次而发生滴度降低，则需要重新制备高滴度储液。如果高滴度储液代数大于P5，则可能有过多的缺陷型干扰颗粒感染细胞，而不能进行适当复制或生成蛋白。如果将已有储液接种后，重新分离得到了新的空斑（DIP不会形成空斑），则该储液可用于制备高滴度储液。

P3病毒储液能否用于生成更多病毒？P4或P5储液呢？

可以，用于制备P2病毒储液的实验方案也可用于制备P3、P4或P5病毒储液。我们不建议制备代数高于P5的储液，因为这会产生更多的缺陷型感染颗粒，并会降低蛋白表达水平。

杆状病毒能否感染哺乳细胞？能否感染果蝇细胞？

可以，杆状病毒可以感染哺乳细胞，但是需要非常高的滴度。杆状病毒在肝细胞中的感染能力最强。但是，只有在使用高滴度储液直接感染细胞时，才有交叉感染风险。

杆状病毒可以感染果蝇细胞，但不能在果蝇细胞中复制。普通杆状病毒系统中驱动目的基因表达的启动子都是晚期启动子，并且需要来自杆状病毒基因组的早期蛋白。因此，它们在S2细胞中无效，因为无法生成早期蛋白。

在12孔板中，1个空斑需要感染多少细胞？

通常，每孔加入含0.5 x 106个细胞的培养基2.5–3 mL，是一个良好的开端。第3天开始发生裂解。在第3-7天（90%细胞死亡），可收获和扩增病毒。

我想对空斑进行PCR，并以相同的空斑制备储液。你们有何建议？

我们的研发团队通常会将病毒加到含0.5 x 106细胞/孔的12孔培养板中，每孔总体积为2.5 mL。大约3天后，取出0.75 mL培养液用于制备PCR用DNA，将剩余培养基收集至Eppendorf管中，作为P1病毒储液。此外，也可以挑选一个空斑，保存在Grace’s培养基中。

感染后，收获高滴度病毒的最佳时间是什么？如果延长细胞感染时间，会发生什么？

我们建议在细胞裂解率达到90%时，收获高滴度病毒。大约需要5-7天。如果延长细胞感染时间，则裂解细胞释放的蛋白酶会降解病毒表面蛋白，导致传染性病毒量降低。

当病毒感染T25或T75培养瓶后，何时开始出现细胞裂解？在某一时间点的细胞裂解率是多少？

这取决于病毒的加入量。如果感染细胞的MOI为5，则细胞通常在24小时被感染，细胞在65小时左右开始裂解。病毒使用量越少，则所需时间越长，反之亦然。

为繁殖得到更多的重组病毒储液，应使用的MOI是多少？应在何时收获病毒？

繁殖病毒储液时，为避免缺陷性干扰颗粒（DIP）的影响，应使用低MOI（0.03–0.1）。低MOI可确保每个细胞的病毒颗粒感染量低于1，防止DIP扩增。当细胞存活率为15%时，是合适的病毒收获时间。

注意：DIP是接近正常的病毒衣壳，含缺陷性基因组，无法进行成功的复制。尽管该“颗粒”本身无感染性，但当它与正常病毒颗粒或一些其他类型的DI颗粒共感染时，可以进行复制。

有什么办法可以使杆状病毒空斑更明显或更突出？

您可使用中性红或MTT对单层细胞进行染色，使空斑更明显。或者，您可延长室温下的空斑形成时间（平均2-5天），增强重组空斑的对比度。但是，中性红空斑染色法不适用于空斑纯化和病毒扩增。

你们建议感染细胞所使用的病毒滴度为多少？

用于表达研究时，我们建议使用滴度为>1 x 108 pfu/mL的病毒储液。

计算病毒滴度的公式是什么？能否举个例子？

请参见以下公式：

pfu/mL =空斑数量(pfu)/稀释系数x接种体积(mL)

例如，如果病毒稀释度为10-8的孔中含有18个白色空斑，则病毒滴度计算为：

X pfu/mL = 18 pfu/10-8 x 1 mL
X = 1.8 x 109 pfu/mL

你们能否概括出空斑实验法的主要步骤以及对该实验的建议？

以下是空斑实验法的主要步骤：
•在6孔板中接种细胞，并使细胞到达80%融合
•对P1病毒储液（1–10-5）进行连续稀释，并加到细胞中
•在27°C孵育1小时
•在培养基中混合1%融化的琼脂糖
•移除病毒上清液
•在细胞上覆盖含琼脂糖的培养基
•将培养板静置2-3小时，使琼脂糖完全凝固
•培养10-14天
•计算空斑数量

进行该实验时，我们建议：
•细胞应处于非常健康的状态，代数较低（10-20代），处于对数生长期，并且存活率高于95%
•确认病毒储液是无菌的（无污染）
•使用高质量、低熔点的琼脂糖
•琼脂糖培养基的温度非常重要——太热，则细胞会死亡；太冷，则琼脂糖凝固太快
•覆盖琼脂糖培养基后等待2-4小时，使琼脂糖达到100%凝固
•在稀释的板中，空斑数量按下述公式计算：

（1/稀释度）x 空斑数量 = pfu/mL

例如，如果在稀释度为10-6的板中形成50个空斑，则为1/(10-6) x 50 = 5 x 107 pfu/mL

如何确定病毒储液的滴度？

我们建议您采用空斑实验法检测病毒储液的滴度。如果需要，您也可以利用空斑实验纯化单个病毒克隆。

使用Bac-N-Blue表达系统获得含插入片段的重组病毒后，可使用哪种引物进行验证？

我们推荐使用以下序列设计正向和反向引物：

正向引物：5´-TTTACTGTTTTCGTAACAGTTTTG-3´ Tm = 62°C
反向引物：5´-CAACAACGCACAGAATCTAGC-3´ Tm = 58°C

这些引物的侧翼为多角体区域，可兼容所有基于多角体启动子的杆状病毒转移载体。利用O'Reilly等（1992年）的命名法，正向PCR引物的结合发生于多角体基因起始点前方的–44 (nt 4049)至–21 (nt 4072)。反向PCR引物的结合发生于多角体基因的+794 (nt 4886)至+774 (nt 4866) 3´。

能否将自己的杆状病毒转移载体与Bac-N-Blue DNA一起使用？

可以，任何含有ORF603和ORF1629序列的杆状病毒转移载体都可使用。

“野生型”表示什么？使用Bac-N-Blue表达系统时，野生型空斑的出现率是多少？

“野生型”表示可产生包涵体阳性（occ+）空斑的、未切割的Bac-N-Blue DNA。野生型空斑的出现率低于20%。

Bac-N-Blue表达系统与Bac-to-Bac表达系统有何区别？

在Bac-N-Blue系统中，转移载体和杆状病毒DNA的重组发生于昆虫细胞中。Bac-N-Blue载体是一种线性AcMNPV衍生物，含有不完整的(3’) lacZ片段。相应的转移载体含有一个5’ lacZ片段。在同源重组的基础上，重组Bac-N-Blue杆状病毒DNA将具有一个受PETL启动子控制的lacZ基因。因此，重组Bac-N-Blue杆状病毒在空斑实验中会形成蓝色空斑，易于识别。在Bac-to-Bac表达系统中，转移载体与杆状病毒DNA间的重组或位点特异性转座发生于大肠杆菌（DH10Bac）中。在Bac-to-Bac表达系统中，含重组杆状病毒DNA的菌落筛选发生于含50 μg/mL卡那霉素（杆状病毒质粒）、7 μg/mL庆大霉素（pFastBac）、10 μg/mL四环素（辅助性质粒）、100 μg/mL Bluo-gal和40 μg/mL IPTG的Luria琼脂板上。

在昆虫细胞中表达重组蛋白，是否有必要加入Kozak序列？

尽管Kozak序列在哺乳细胞翻译起始中的重要性已被证明，但昆虫细胞中是否也严格遵循Kozak规则仍有争议。确定其重要性的唯一方法是，对相同蛋白从不同起始序列的表达进行直接对比。即使这样，一种蛋白实现最佳表达所使用的规则可能并不适合其它蛋白。以下四篇文献证明了Kozak序列对昆虫细胞中的表达效率无任何影响：

•Hills D, Crane-Robinson C (1995) Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression. Biochim Biophys Acta 1260(1):14–20.
•Ranjan A, Hasnain SE (1995) Influence of codon usage and translational initiation codon context in the AcNPV-based expression system: computer analysis using homologous and heterologous genes. Virus Genes 9(2):149–153.

为了实现共表达，如共表达一种蛋白的两个亚基，能否使用两种不同的重组病毒共感染昆虫细胞？

可以。多种五亚基蛋白，如人类复制因子C，已使用重组杆状病毒进行表达。为实现最佳的多亚基蛋白表达，我们建议生产出每个亚基的、独立的高滴度储液（HTS）。利用这种方法，可通过改变每个亚基HTS的感染复数，控制每个亚基的表达量。请参考以下文献，获取更多信息：

•Chen W and Bhal OP (1991) Recombinant carbohydrate and selnomethionyl variants of human choriogonadotropin. J Biol Chem 266(13):8192–8197.
•Chen WY and Bhal OP (1991) Selenomethionyl analog of recombinant human choriogonadotropin. J Biol Chem 266(15):9355–9358.
•Fabian JR, Kimball SR, Jefferson LS (1998) Reconstitution and purification of eukaryotic initiation factor 2B (eIF2B) expressed in Sf21 insect cells. Protein Expr Purif 13(1):16–22.

什么是感染复数？如何计算？

MOI或感染复数，是指某个实验中，感染单个细胞的平均病毒颗粒数量。您可使用以下公式计算MOI：

MOI (空斑形成单位(pfu)/细胞) = [滴度(pfu) x 接种物所用病毒储液体积(mL)] / [细胞密度(细胞/mL) x 培养物体积(mL)]

能否使用杆状病毒表达系统进行重组蛋白的放大生产？如果可以，应使用哪种方法？

可以，可以进行大规模表达实验。下表描述了不同大规模实验方法、要求、优势和参考文献：

- Stirred bioreactor
- Airlift fermentor
- Insect larvae

我应如何保存病毒储液？

如果培养基是无血清的，可加入血清至浓度为10%。血清蛋白可作为蛋白酶的底物，防止病毒外壳蛋白发生降解。将病毒储液置于4°C避光保存。分装储液可保存在–80°C，使用前应检测病毒滴度，因为冻融循环会导致病毒滴度降低10-100倍。

我是否需要纯化重组病毒，除去未切割或非重组的病毒DNA？

需要。重组DNA存在未切割（occ+）DNA污染，将导致重组病毒随时间而逐渐稀释，因为未切割（野生型，occ+）病毒的感染和复制效率高于重组病毒。同时，使用纯化的、单一病毒群体进行起始表达，将确保得到可重复的结果。

病毒感染早期和末期有哪些形态改变？

请查看以下关于病毒感染不同阶段的描述：

早期
细胞直径增加——细胞直径可能增加25–50%。
细胞核体积增加——细胞核可能“填满”细胞。

末期
细胞生长停止——与单纯的细胞对照相比，细胞停止生长。
颗粒状外观
病毒出芽迹象——细胞出现泡状外观
病毒包涵体——少量细胞会含有包涵体，表现为昆虫细胞核内出现折射晶体。
脱落——细胞从培养皿或培养瓶上脱落。
晚期的细胞裂解——少量细胞可能会充满包涵体病毒、发生死亡和裂解，留下单层清理迹象。

如何证明已成功转染并生产杆状病毒？

贴壁Sf9细胞呈聚集状，接触点较小。感染后Sf9细胞聚集悬浮于培养液中，细胞体积变大。

对于杆状病毒表达系统的转染，有哪些一般性建议？

请遵循以下建议：

•细胞应处于非常健康的状态，代数较低（5-15代），处于对数生长期，并且存活率高于95%。
•DNA必须高度纯化，无内毒素
•转染时，不可使用抗生素
•Cellfectin试剂应完全重悬
•设置对照组（培养基对照、DNA对照和转染试剂对照），用于对比和问题排查

在昆虫细胞中接种重组病毒后，何时可以收获蛋白？

昆虫细胞中的蛋白表达高峰，取决于感染复数（MOI）、感染时间和目标蛋白。系统优化包括使用MOI为5-10，表达时间为48-72小时。72小时后表达的蛋白可能是经过异常加工的，因为较高的病毒载量可引起细胞进程故障。

杆状病毒有利于表达毒性蛋白？

是的，杆状病毒是表达毒性蛋白（即，膜蛋白）的良好候选方案。多角体启动子在感染后18-24小时才能表达至最高水平。多角体启动子在裂解晚期激活。也就是说，其在早期8小时的时候活性极低，所以，如果目的基因毒性很强，可能会造成问题。该问题的解决方法是转换到诱导型表达系统。跨膜蛋白通常难以在任何系统中表达。

I cannot grow this white colony in liquid culture. What should I do?

The concentration of gentamicin might be too high. Try lowering the amount to 5 µg/mL and try adding more of the colony to the culture medium.

What has happened when I see blue colonies? How about colonies which are blue in the center and white on the edges?

In the case of a blue colony, the E. coli has the bacmid and the plasmid in it, allowing the cells to survive the selection process. However, because the transposition has not occurred, the LacZ gene is not disrupted. For bulls-eye colonies, this indicates that the transposition took place when the colony was growing. Re-streaking for an isolated clone from the white portion of the mixed colony should yield some colonies where transposition occurred.

I'm getting mostly white/wild-type plaques instead of blue/recombinant plaques. What am I doing wrong?

This is typically an indication of poor homologous recombination. Check the plasmid/linear DNA ratio you used. If there are some blue plaques, however, expand those viruses and check for their protein. In our experience, they are correct, even if they were in relatively low abundance.

I've infected my cells and see large polyhedra in one cell and smaller polyhedra (more numerous) in a neighboring cell. Is this normal?

Yes, cells are infected with wild-type virus individually and will develop polyhedra at different rates until all the cells in the flask are infected. The polyhedra in cells will form in approximately 3-4 days, differing in size and number until they reach their maximum capacity and burst the cell, releasing tiny particles of virus into the medium.

I'm worried that I am not getting plaques. How many days does it take to see plaques and what size are they typically?

Normally, very small white dots show up about 5-7 days and 1 mm plaques show up around day 10. Plaques can vary in size from 1 mm to 4 mm.

I forgot to add Bluo-gal to my plates. Can I add it later?

On the day you intend to pick plaques, make a solution of Bluo-gal in DMSO at 20 mg/mL. Add 50 µL per plate and spread with a glass spreader under sterile conditions. Wait 30-60 min, and your plaques should turn blue.

What might cause plates to turn blue along with blue plaques?

There are a few things that can turn plates blue:

- Too much virus when plating. Try a higher dilution.
- Cells are being singed when plated with hot melted agarose. This lyses the cells and releases lacZ into the agarose, turning it blue. Double-check plating temperatures. If plates are too wet, the blue can diffuse.

After addition of the agarose overlay to my cells, they look like crescents or are granular. What happened?

The agarose overlay was too hot. After addition of the agarose overlay, cells should still be round and healthy.

My agarose overlay is floating. Is this because I did not aspirate the medium completely?

Yes, this is indicative of an aspirating problem on the plaques. The agarose overlays were “floating” because the medium was not completely aspirated from the plates. The plates need to be completely dry before the agarose is placed over the cells, especially when plaques will be picked. To do this, we typically tip the plate slightly and keep going around the rim of the plate with the Pasteur pipette tip, being careful not to disturb the cell monolayer. If any medium pooling at the rims of the plates (they will be small pools) is seen, continue to aspirate. This “floating” agarose overlay problem may also result in wild-type contamination. The wild-type virus is able to migrate to other portions of the plates and contaminate recombinant plaques. Wild-type virus replicates much faster than recombinant virus, and can quickly overwhelm the recombinant virus.

I see what looks like small plaques, but they are too difficult to visualize. What could be the problem?

Too many cells were seeded; we recommend seeding 8 x 10e5 cells per well for a 6-well plate.

I think I've added too much virus. How important is the MOI? What happens if too much virus is used?

An MOI of 5-10 is typically used. If too much virus is added, unfortunately the cells die too soon and the protein expression level goes down.

I'm yielding no plaques from my baculovirus plaque assay. What are the possible causes for this?

The kinetics of infection may be slower than expected. Observe plates until the 8-9th day after infection. If no plaques appear, investigate the following:

- If the cells are not healthy, then poor-quality or no plaques can result. Ideally, cells should be in mid-log phase and have a viability of greater than 90%. Cells should double at least once before infection stops growth. Ensure that the correct amount of cells was used at ~70% confluency.
- The viral replication cycle can be inhibited due to poor nutritional and physical conditions of the cell.
- The temperature of the agarose is also crucial. After overlaying the agarose, the plates should be left untouched for 1 hour for the agarose to completely solidify.
- Excessive condensation during incubation at 27 degrees C can inhibit plaque formation-remove paper towels or open the container containing plates as soon as condensation appears.
- The viral titer is too low: Use a higher viral titer. You may need to re-infect your cells and collect a higher titer of your viral stock.

I performed PCR analysis of recombinant virus from several plaques and am seeing a mix of wild-type and recombinant virus using the Bac-N-Blue expression system. What should I do?

If there is contamination with wild-type virus in all of your samples, take a P1 viral stock and redo the plaque assay. Be sure to select well-spaced, occ- (occlusion body negative) plaques. If you are having difficulty distinguishing a recombinant plaque from a non-recombinant plaque, try using one of the pBlueBac vectors. Recombinant plaques will be blue when chromogenic substrate is incorporated into the medium during the agarose overlay (see page 14 of the manual for more information).

It has been 4 days post-infection, and I do not see signs of viral infection with my Bac-N-Blue expression system. What should I do?

Please compare your cells-only plate to the infected plate. The uninfected cells should appear overgrown when compared to infected cells, as transfection inhibits growth. If this is seen, keep checking the infected cells daily for other signs of infection (nuclear swelling, detachment from the plate, viral budding, and lysis). The kinetics of infection may be slower than expected. If cell growth does not appear to be inhibited, you may consider the following factors:

- How was the DNA prepared? We recommend using a resin-based DNA purification system, such as our PureLink HiPure Plasmid Prep Kits.
- Were cells in log phase? What was their confluency? We recommend that cells be in log phase, 95% viable, and plated at 50-70% confluency for successful transfection.
- What transfection reagent was used? We recommend using Cellfectin II Reagent.


Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I cannot detect any recombinant fusion protein after using the BaculoDirect Expression Kit. What could be the cause for this and what do you suggest I try?

Please check the construction of your entry clone, and ensure that the insert is in frame with the vector. Analyze the recombinant viral DNA by PCR to confirm the correct size and orientation of your insert after the LR reaction. Sequence your PCR product to verify the proper reading frame for expression of the epitope tag.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm getting a low-titer P1 viral stock and would like to generate a high-titer stock. What should I do?

To get a high-titer stock, reinfect cells with the P1 stock and generate a P2 high-titer stock. Follow the directions in the BaculoDirect manual on page 18 to generate your P2 stock.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I performed an LR reaction, followed by transfection into Sf9/Sf21 insect cells, but do not see any signs of infection even though it's been 72 hours. What should i do?

Please see our recommendations below:

- Check the LR reaction by PCR analysis prior to transfection into insect cells.
- We recommend using Grace's Insect Cell Culture Medium, Unsupplemented during the transfection experiment instead of serum-free medium, as components in serum-free medium may interfere with transfection.
- Ensure that FBS, supplements, or antibiotics are not included during transfection, as the proteins in these materials can interfere with the Cellfectin II Reagent.
- Use the LR recombination reaction using the pENTR/CAT plasmid as a positive control and Cellfectin II Reagent only (mock transfection) as a negative control.
- Ensure that cells are in the log phase of growth with >95% viability, and the amount of cells are in accordance with the suggestions in the manual.
- Cells may not show signs of viral infection for up to a week depending on transfection efficiency; continue culturing and monitor cells daily for signs of infection.

I see a precipitate in my ganciclovir solution. What can I do?

Warm the ganciclovir solution in a water bath at 37 degrees C for 5-10 min, then vortex for a few minutes. The precipitate should go back into solution.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I am purifying my secreted protein expressed in insect cells with a His-tagged purification column and getting no yield of my protein. Why and what can I do?

Media used to culture insect cells usually have an acidic pH (6.0-6.5) or contain electron-donating groups that can prevent binding of the 6xHis-tagged protein to Ni-NTA. Amino acids such as glutamine, glycine, or histidine are present at significantly higher concentrations in media for growing insect cells than in media for growing mammalian cells, and compete with the 6xHis-tagged protein for binding sites on Ni-NTA matrices. Grace's medium (Thermo Fisher Scientific), for example, contains approximately 10 mM glutamine, 10 mM glycine, and 15 mM histidine.

Dialysis of the medium against a buffer with the appropriate composition and pH (8.0) similar to the lysis buffer recommended for purification under native conditions usually restores optimal binding conditions. Note that depending on the medium used, a white precipitate (probably made up of insoluble salts) can occur, but normally the 6xHis-tagged protein remains in solution. This can be tested by either protein quantitation if using a protein-free medium or by monitoring the amount of 6xHis-tagged protein by western-blot analysis. After centrifugation, 6xHis-tagged protein can be directly purified from the cleared supernatant.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

My viral stock used to work well from my Bac-to-Bac baculovirus expression system, but after a couple of months, there is much less protein expression. Why is this?

Please review the following possibilities and solutions:

- Viral stock was amplified using high MOI originally: Go back to the lower-passage viral stock and do a low-MOI amplification.
- Did not spin down and get rid of cells when harvesting viral supernatant: Go back to the lower-passage viral stock and do a low-MOI amplification; if this viral stock is P2, this stock can be used in amplification.
- For some genes, the virus can become very unstable: Free the aliquoted P2 viral stock and do one run of amplification after reviving.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm getting low protein yield when using the Bac-to-Bac baculovirus expression system. What are the possible reasons for this?

Low protein yield may occur due to the following reasons:

- Viral stock contains a mixture of recombinant and non-recombinant baculovirus: Perform plaque purification to isolate recombinant baculovirus.
- Baculovirus is not recombinant: Verify transposition by PCR analysis of bacmid DNA using pUC/M13 forward and reverse primers; re-transfect insect cells with new recombinant bacmid DNA.
- Use too low or too high viral titer: Vary the MOI.
- Time of cell harvest is not optimal: perform a time course of expression to determine the optimal time to obtain maximal protein expression.
- Cell growth conditions and medium are not optimal: Optimize cell culture conditions based on the size of your culture vessel and expression conditions; we recommend using Sf-900 II SFM or Sf-900 III SFM for optimal cell growth and protein expression.
- Cell line is not optimal; try other insect cell lines.
- Cells were harvested too late: Do a time-course experiment and harvest cells at different time points.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I don't see any signs of cell infection 72 hours after infection with my P2 viral stock. Why is this?

Check the MOI. It may be low because the titer of the P1 virus is lower than what was estimated.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Using the Bac-to-Bac baculovirus expression system, I've transduced my cells but do not see any signs of viral production. It has been 5 days. What happened and what should I do?

Please see the possible reasons and suggestions below:

- Mixture of Cellfectin II Reagent and bacmid was not performed or was not incubated long enough: Mix the Cellfectin II Reagent and bacmid well by tapping or gentle vortexing, and incubate the mixture for 15-45 min.
- Bacmid yield is lower than estimated: Set up an optimization with different amounts of bacmid.
- Bacmid is sheared during purification or freeze/thaw: Verify the integrity of bacmid on a gel.
- Incubation time is not long enough: Incubate mix for 8 hr at 27 degrees C.
- Cells used are of high passages or have passed log-phase growth: For best results, use cells between 8-15 passages; plate cells when they are in log-phase growth.
- Cellfectin II Reagent has been frozen: Purchase a new vial.
- Medium used contains serum: Use unsupplemented Grace's medium in transfection.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Both transfected cells and Cellfectin II Reagent alone negative control cells are dead within 36 hours. What could cause this?

There are several possibilities:

- Using media containing antibiotics during transfections.
- Plating cells at too low a density: We recommend at least 70% confluence.
- Using cells at too early a passage: We recommend growing cells for at least 5 passages before using them for transfection.
- Contamination because of no pen/strep after the transfection: After 5-8 hr incubation with the transfection mixture, remove the mixture and add antibiotics containing media/well.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

My transfected cells are floating up and look dead, but the Cellfectin II Reagent alone control plate is fine. What could be happening?

This may be due to contamination or cytotoxicity from the bacmid prep. Make sure to include a negative control that is the bacmid only without Cellfectin II Reagent. Additionally, use the PureLink HiPure plasmid prep kit, not the silica-based miniprep kit for bacmid prep.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can an old low-titer stock be used to make a high-titer stock?

If this lower-titer stock is a P1 or P2 stock, a viral amplification protocol can be used. If the low-titer stock was once a high-titer stock, but has dropped titer due to age or the stock was propagated many generations, then it may be necessary to regenerate the high-titer stock. If the high titer stock is >P5, then there may be an excessive amount of defective interfering particles that infect cells but do not properly replicate or produce protein. If the existing stock is plated out and a fresh plaque is re-isolated (DIPs do not form plaques), a new high-titer stock can be established.

Can a P3 viral stock be used to generate more viruses? How about a P4 or P5 stock?

Yes, the same protocol used to make your P2 viral stock can be used to make a P3, P4, or P5 viral stock. We don't recommend making the stock higher than P5, as more defective interfering particles will be produced and a decrease in protein expression level will occur.

Can the bacluovirus infect mammalian cells? How about Drosophila cells?

Yes, baculovirus can infect mammalian cells, although only at very high titers. Baculovirus works best in liver cells. However, there is no danger of cross-contamination unless the cells are directly infected with the high-titer stocks. Bacuolvirus can infect Drosophila cells; however, it will not replicate in these cells. The promoters used to drive expression of your gene in a typical baculovirus system are both late promoters and require earlier proteins from the baculovirus genome. Thus, they will not work in S2 cells since the early proteins are not made.

How many cells need to be infected in a 12-well plate with 1 plaque?

Typically, 0.5 x 106 cells per well in 2.5-3 mL is a good starting point. Lysis should begin by day 3. Virus may be harvested and amplified between 3 and 7 days (90% cell death).

I'd like to perform PCR on a plaque, and also make a stock from the same plaque. How do you suggest I do this?

Our R&D team will typically pick a plug and add it to a 12-well dish with 0.5 x 10e6 cells/well and 2.5 mL total volume per well. After approximately 3 days, remove 0.75 mL to make DNA for PCR and keep the remaining medium in an Eppendorf tube as your P1 viral stock. As an aside, it is okay to pick a plaque and store it in Grace's medium.

What is the optimal time for harvesting high-titer virus following infection? What happens if I let the cells go longer?

We recommend harvesting high-titer virus when there is 90% cell lysis. This takes approximately 5-7 days. If the cells go longer, the proteases released from the lysed cells will start to degrade viral surface proteins and result in less infectious virus.

After infecting T25 or T75 flasks with virus, when do you begin to see cell lysis and what percentage of cells will be lysed at certain time points?

This is dependent on how much virus is added. If cells are infected at an MOI of 5, usually cells are infected at 24 hours, and cells begin to lyse at around 65 hours. If less virus is used, this takes longer, and more virus takes less time.

To propagate more recombinant virus stock, what MOI should I use and when should I harvest the virus?

When propagating virus stock, use a low MOI (0.03-0.1) in order to avoid effects of defective interfering particles (DIPs). A low MOI, which ensures no more than 1 virion per cell, prevents the amplification of DIPs. A harvest time based on 15% cell viability is appropriate. NOTE: DIPs are nearly normal virus capsids containing genomes that are defective and are unable to undergo successful replication. While this "particle" is not infectious by itself, it can replicate when co-infected with normal virion, or with some other types of DI particles.

Is there a way to make baculovirus plaques more visible or distinctive?

You can stain the monolayer with neutral red or MTT to make the plaques more visible. Alternatively, you can allow the plates to develop for a few days longer (2-5 days on average) at room temperature to increase the contrast in recombinant plaques. However, the plaques stained with neutral red cannot be used for plaque purification and viral amplification.

What viral titer do you suggest having to infect my cells?

We suggest using a viral stock with a titer of >1 x 10e8 pfu/mL for expression studies.

What is the equation to calculate viral titer? Do you have an example I can use?

Please see the equation below:
pfu/mL = number of plaques (pfu)/dilution factor x mL of inocula
So, if you have a well with viral dilution of 10-8 containing 18 white plaques, the viral titer is calculated as followed:
X pfu/mL = 18 pfu/10-8 x 1 mL
X = 1.8 x 10e9 pfu/mL

Can you outline the main steps of performing a plaque assay, and any suggestions when performing this assay?

Please see the method below for an outline of the main steps of performing a plaque assay:

- Plate cells at 80% confluency in a 6-well plate
- Make a serial dilution of the P1 viral stock (1-10-5) and add to cells
- Incubate for an hour at 27 degrees C
- Mix 1% melted agarose into the medium
- Remove the viral supernatant
- Overlay the cells with the medium containing agarose
- Leave the plates for 2-3 hours for agar to completely solidify
- Incubate plates for 10-14 days
- Count plaques

When performing this assay, we suggest:

- Use cells that are in excellent health, of low passage (10-20) in log-phase growth, and high viability (>95%)
- Check viral stock for sterility (free of contamination)
- Use high-quality, low melting point agarose
- The temperature of the medium with agarose is crucial-too hot, cells will die; but if too cold, it will solidify too quickly
- Wait 2-4 hours before removing the plate after overlay so that the agarose can 100% solidify
- Count plaques on a dilution plate where (1/dilution) x # of plaques = pfu/mL
e.g., if you have 50 plaques on the 10-6 plate, then you have 1(10-6) x 50 = 5 x 10e7 pfu/mL

How do I determine the titer of my viral stock?

We recommend you perform a plaque assay to determine the titer of your viral stock. You may also perform a plaque assay to purify a single viral clone, if desired.

What primers can I use to verify recombinant viruses containing inserts with my Bac-N-Blue expression system?

We recommend designing the forward and reverse primers with the following sequences:

- Forward primer: 5'-TTTACTGTTTTCGTAACAGTTTTG-3' Tm = 62 degrees C
- Reverse primer: 5'-CAACAACGCACAGAATCTAGC-3' Tm = 58 degrees C

These primers flank the polyhedron region and are compatible with all polyhedron promoter-based baculovirus transfer vectors. The forward PCR primer binds from -44 (nt 4049) to -21 (nt 4072) in front of the start of the polyhedron gene, using the nomenclature of O'Reilly et al., 1992. The reverse PCR primer binds at +794 (nt 4886) to +774 (nt 4866) 3' of the polyhedron gene.

Can I use my own baculovirus transfer vector with the Bac-N-Blue DNA?

Yes, any baculovirus transfer vector can be used so long as it contains ORF603 and ORF1629 sequences.

What does "wild-type" refer to and what is the frequency of wild-type plaques I should except when using the Bac-N-Blue expression system?

“Wild-type” refers to uncut Bac-N-Blue DNA that will produce occlusion body positive (occ+) plaques. The frequency of wild-type plaques is less than 20%.

What is the difference between the Bac-N-Blue expression system and the Bac-to-Bac expression system?

In the Bac-N-Blue system, recombination between the transfer vector and the baculovirus DNA occurs in insect cells. The Bac-N-Blue vector is a linearized AcMNPV derivative that contains an incomplete (3') lacZ fragment. The corresponding transfer vector contains a 5' lacZ fragment. Upon homologous recombination, the recombinant Bac-N-Blue baculovirus DNA will have a complete lacZ gene that is under the control of the PETL promoter. Thus, recombinant Bac-N-Blue baculovirus will provide blue plaques in the plaque assay and can be easily identified. In the Bac-to-Bac expression system, recombination or site-specific transposition between transfer and baculovirus DNA occurs in E. coli (DH10Bac). In the Bac-to-Bac expression system, selection of colonies containing recombinant baculovirus DNA occurs in the presence of Luria Agar plates with 50 µg/mL kanamycin (bacmid), 7 µg/mL gentamycin (pFastBac), 10 µg/mL tetracycline (helper plasmid), 100 µg/mL Bluo-gal, and 40 µg/mL IPTG.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Do I need to include a Kozak sequence for expression of recombinant proteins in insect cells?

While the importance of a Kozak consensus sequence in translation initiation has been demonstrated in mammalian cells, there seems to be some debate as to whether the Kozak rules are as stringent in insect cells. The only way to determine its importance would be a direct comparison of expression of the same protein from different initiation sequences. Even then, the rules for optimal expression of one protein may not hold for another. Here are two references which indicate that a Kozak consensus sequence does not have any effect on efficiency of expression in insect cells:

- Hills D, Crane-Robinson C (1995) Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression.
- Biochim Biophys Acta 1260(1):14-20.
- Ranjan A, Hasnain SE (1995) Influence of codon usage and translational initiation codon context in the AcNPV-based expression system: computer analysis using homologous and heterologous genes. Virus Genes 9(2):149-153.

Is it possible to co-infect insect cells with two different recombinant viruses in order to co-express, for example, two subunits of a protein?

Yes, it is possible. Several five-subunit proteins, such as human replication factor C, have been expressed using recombinant baculovirus. We recommend that a separate high-titer stock (HTS) of each subunit be produced to optimally express the multi-subunit protein. This way, the amount of each subunit expressed can be controlled by varying the multiplicity of infection (MOI) of each subunit's HTS. Please refer to the following articles for more information:

- Chen W and Bhal OP (1991) Recombinant carbohydrate and selnomethionyl variants of human choriogonadotropin. J Biol Chem 266(13):8192-8197.
- Chen WY and Bhal OP (1991) Selenomethionyl analog of recombinant human choriogonadotropin. J Biol Chem 266(15):9355-9358.
- Fabian JR, Kimball SR, Jefferson LS (1998) Reconstitution and purification of eukaryotic initiation factor 2B (eIF2B) expressed in Sf21 insect cells. Protein Expr Purif 13(1):16-22.

What is the multiplicity of infection, and how can I calculate it?

The MOI, or multiplicity of infection, is the average number of viral particles that infect a single cell in a specific experiment. You can calculate the MOI with the following equation:
MOI (pfu/cell) = [titer (pfu) x viral stock volume (mL) used in inocula] / [cell density (cells/mL) x culture volume (mL)]

Can I scale-up the production of recombinant protein using the baculovirus expression system? If so, what methods are available?

Yes, large-scale expression experiments can be performed. Please see below for different large-scale methods, requirements, added benefits, and references:
- Stirred bioreactor
- Airlift fermentor
- Insect larvae

How can I store my viral stock?

If the medium is serum-free, add serum to 10%. Serum proteins act as substrates for proteases and therefore prevent degradation of viral coat proteins. Store viral stocks at 4 degrees C, and protect from light. Aliquots can be stored at -80 degrees C, but viral titer should be checked before use, as freeze/thaw cycles of the virus can result in a 10- to 100-fold decrease in viral titer.

Do I need to purify my recombinant virus away from an uncut or non-recombinant viral DNA?

Yes. Contamination of your recombinant DNA with uncut occlusion body positive (occ+) DNA will lead to dilution of your recombinant virus over time because, in general, uncut (wild-type, occ+) virus infects and replicates at higher efficiency than recombinant virus. Also, initiating expression studies with a pure, single virus population will ensure reproducible results.

What does viral infection look like in early, late, and very late stages?

Please see the description below of the different stages of viral infection:

Early
- Increased cell diameter-a 25-50% increase in the diameter of the cells may be observed.
- Increased size of cell nuclei-the nuclei may appear to "fill" the cells.

Late
- Cessation of cell growth-cells appear to stop growing when compared to a cell-only control.
- Granular appearance
- Signs of viral budding-vesicular appearance of cells.
- Viral occlusions-few cells will contain occlusion bodies, which appear as refractive crystals in the nucleus of the insect cell.
- Detachment-cells release from the dish or flask.

Very late
- Cell lysis-a few cells may fill with occluded virus, die, and burst, leaving signs of clearing in the monolayer.

What should I look for to indicate a successful transfection in which baculovirus is produced?

Adherent Sf9 cells round up and show a smaller contact point. Infected Sf9 cells in suspension culture round up and look larger when infected.

What are some general suggestions for transfection in baculovirus expression systems?

Please follow the recommendations below:

- Cells should be in excellent health, of their low passages (5-15), in log-phase growth, with viability >95%
- DNA must be of high purity, free of endotoxin
- No antibiotics should be used during transfection
- Cellfectin reagent has to be completely resuspended
- Include controls (media control, DNA control, and transfection reagent control) for comparison and troubleshooting

When should I harvest my protein after I have inoculated my insect cells with recombinant virus?

Peak expression of protein in insect cells is dependent on the multiplicity of infection (MOI), expression time, and the protein being expressed. Guidelines to optimize your system include using an MOI of 5-10 and an expression time of 48-72 hours. Protein expressed at times later than 72 hours may be processed aberrantly, because the large virus load can cause a breakdown of cellular processes.

Is baculovirus good for expressing toxic proteins?

Yes, baculovirus is a good candidate for the problem of expressing toxic proteins (i.e., membrane proteins). The polyhedron promoter does not express at maximal levels until 18-24 hr after infection. The polyhedron promoter is active late in the lytic cycle. That being said, it is minimally active as early as 8 hours, so if the gene is very toxic, there may be a problem. The solution in that case would be to switch to an inducible expression system. Transmembrane proteins can often be difficult to express in any system.