CN1726290A - 产生用于筛选活性物质的经遗传修饰生物的方法 - Google Patents
产生用于筛选活性物质的经遗传修饰生物的方法 Download PDFInfo
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Abstract
本发明涉及产生用于筛选活性物质的经遗传修饰生物的方法，该方法包括步骤：a)通过该生物的遗传修饰诱导异源表达至少一种蛋白质或蛋白质片段；b)分析修饰基因表达模式并鉴定已被代偿性和差异调节的基因；和c)该生物的显型。本发明还涉及通过所述方法可以得到的经遗传修饰生物。
Description
公知异源表达靶蛋白的遗传修饰的酵母被用于药物筛选，该靶蛋白将被所测试的物质抑制。在本发明的范围内，异源表达指表达与该生物无关的基因，或者以改变的表达模式表达该生物的内源基因，尤其表达被增强或减弱和/或表达在时间和/或空间(例如，在其他区室中，例如在高等生物的其他组织中)上发生改变。最简单的例子是，异源表达导致酵母的可检测的修饰表型，通常为生长抑制。在本发明的范围内，生长抑制指增殖速度减慢和/或生长大小减小，还包括细胞死亡(凋亡或坏死)。生长抑制发生的类型还取决于该生物，因而，在酵母中，可以观察到增殖抑制或者裂解，而在最初来自多细胞生物的真核细胞中有时也可观察到调亡。如果异源表达导致该生物的行为和/或形态的改变，该改变可以从外部观察(即，表型改变)，那么该经遗传修饰生物可容易地用于药物筛选，基于物质消除或减弱该表型(例如生长抑制)的能力可以确定测试物质的效能。在将生长抑制作为修饰表型的酵母体系的实例中，优选通过简单生长试验实施效能测定，该简单生长试验也适于高通量筛选(HTS)。与未经遗传修饰生物或者不表达该异源蛋白或蛋白片段的生物相比，遗传修饰生物的可从外部观察的任何改变(形状、大小，等等)或者其行为(生长、细胞分裂速度，等等)的任何改变都被称为表型改变。因而显型(phenotyping)指造成这种改变。
然而，现有技术的该方法有缺点：仅仅小部分异源表达的基因产生可用于药物筛选的经遗传修饰生物的表型。因此，可以设想例如，所有异源表达的激酶中仅有约20-30％在酵母中导致可用于药物筛选的生长抑制。对于剩下的70-80％，生长抑制如此低以至于其不能用于筛选(与对照相比太小的差异导致高背景，从而导致大量假阳性)或者根本不存在生长抑制。
因此，需要产生用于药物筛选的遗传改造的生物的方法，该方法没有现有技术的缺点，并且尤其适于使得某些异源表达的基因也可用于药物筛选，尤其是HTS，这些异源表达的基因在它们所异源表达的生物中不产生任何表型或者不产生任何可用于筛选的表型。
根据本发明，通过产生用于药物筛选的经遗传修饰生物实现了该目的，该方法包括步骤：
a)通过该生物的遗传修饰导致异源表达至少一种蛋白质或蛋白质片段。
b)优选接着测定该遗传修饰生物的表型。
c)分析改变的基因表达模式并鉴定代偿性的差异调节基因。
d)该生物的显型(优选结合异源表达的蛋白质或蛋白质片段，通过缺失、诱变或过表达代偿性调节的基因以增强或产生一种表型)。
本发明基于本发明人的发现：多数基因的异源表达的可检测表型的缺乏是基于这一事实，即作为对异源表达的蛋白质或蛋白质片段的表达的应答，经遗传修饰生物上调或下调(即代偿性差异调节)一些基因的表达。在该情况中，差异调节指与经遗传修饰生物中的调节不同或者异源表达的蛋白质或蛋白片段没有被异源表达。代偿性指差别基因调节是对蛋白质或蛋白质片段异源表达的应答。
本发明使得可能在与简单生化模型相比，本发明使在细胞模型(优选酵母)中发展平台技术成为可能。使用该测定系统，例如可能从化学文库、CombiChem文库和从天然物质的提取物中鉴定抑制剂。该测定系统适于96孔、384孔或1536孔板或者细胞测试的其他常见形式。所要选择的形式部分取决于所选的生物，该选择在技术人员的能力内。
本发明的方法尤其适于某些基因和蛋白质或蛋白质片段，与没有经遗传修饰生物或者不异源表达所述蛋白质或蛋白片段的生物相比，它们在所需生物中的异源表达不导致表型的任何可检测的修饰。可能的是，例如测定蛋白质激酶以及导致转录应答的其他基因产物。然而，其还可能应用于可检测的修饰表型，尤其如果所修饰的表型尽管是可检测的，但是由于特定原因而不适宜用于药物筛选。可以以如此方式通过显型或修饰增强所述表型，从而使该表型可用于药物筛选。因此，在本发明的范围内，显型指在异源表达蛋白质或蛋白质片段的经遗传修饰生物中导致或增强表型，该表型可区别于不异源表达蛋白质或蛋白质片段的生物或者没有经遗传修饰生物。
适宜的生物优选为细胞，这里为真核细胞以及原核细胞，或者适于药物筛选的多细胞非人生物，例如，果蝇(Drosophila)，并优选秀丽线虫(C.elegans)。适宜的真核细胞优选为培养的细胞系，其最初得自多细胞生物(例如，3T3、CHO、HeLa)或者其他的或真核单细胞生物，尤其是酵母。酵母中尤其适宜的又是酿酒酵母(S.cerevisiae)或裂殖酵母(S.pombe)。技术人员非常熟悉酵母细胞的适宜的实验室株系或适宜的真核细胞系。
适宜的蛋白质和蛋白质片段原则上是所有那些在生物中的异源表达导致内源基因的表达模式改变的蛋白质和蛋白质片段。优选所有与发现新的活性物质有关的蛋白质和蛋白质片段，本发明范围内尤其优选激酶、磷酸酶、GPCRs、(尤其小的)GTP酶、蛋白酶和离子通道。
在本发明范围内，术语“药物筛选”包括使用至少一种经遗传修饰生物对一些物质的任一类型的寻找，这些物质作用于一种或多种特定靶基因和/或靶蛋白的活性。原则上，任一类型的物质在这里都是适宜的，例如，任一类型的天然物质(即天然产生的分子，尤其生物分子)以及非天然发生、合成产生的化学物质和从天然物质，尤其生物分子(例如，修饰的肽或寡核苷酸)衍生的物质/衍生物。
异源表达包括通过例如导入适宜的表达载体来向生物导入外来基因或者修饰生物内源基因的表达。因此所需要的遗传修饰可以涉及修饰生物的基因组(例如，通过将稳定载体整合到基因组或者通过各种类型的诱变)，可以是附加体的或者可以仅包括导入适宜的载体，该载体需要通过一种或多种选择标记的恒定选择以便在生物中保留。最适宜的类型取决于多种因素，还取决于生物的类型，并且可以通过熟练技术人员容易地确定。
异源表达涉及至少一种蛋白质或蛋白质片段，但是也可以涉及多种蛋白质或蛋白质片段。可以通过适宜的方法(PCR、RNA印迹、蛋白质印迹等)便利地验证异源蛋白质/片段的表达，之后将经遗传修饰生物的基因表达模式与缺少所述异源蛋白的表达的生物比较，并分析。该分析通过适宜的措施进行，这些措施是技术人员足够熟悉的，使用阵列(优选DNA/RNA或蛋白质微阵列(microarray)或者芯片系统尤其适于该目的。通过比较对照生物(例如，野生型生物或者仅导入空载体的生物，或者对于可诱导的系统，异源基因的表达没有被诱导的经遗传修饰生物)和表达异源基因的经遗传修饰生物的表达模式。与对照生物的表达模式相比，表现出升高或降低程度、或者根本不存在于遗传修饰生物的表达模式中的基因产物被认为是代偿性差异调节的基因，并且可用于显型所述经遗传修饰生物。
显型指在经遗传修饰生物中导致或增强与野生型生物可以区别的表型(或者对于可诱导的系统，仅由异源表达蛋白质或蛋白质片段的经遗传修饰生物产生，并且在不表达该蛋白质或蛋白质片段的所述生物未诱导状态中不产生的表型)，优选该表型适于HTS药物筛选中的评估。所述导致或增强可以在例如减少或消除一种或多种代偿性上调的基因(这可以例如，通过基因组敲除一个或多个代偿性差异调节的基因或者通过诱变发生)或者增强一种或多种代偿下调的基因的表达上发生(这可以通过，例如，使用适宜的表达载体异源表达一种或多种代偿性差异下调的基因实施)。这样，可能产生生物内源的并且由异源表达基因导致的表型，该表型由于一种或多种基因的代偿性差异调节而被阻止(优选生长抑制，但是，尤其在多细胞生物中，其他表型也可能存在于此处)。
另一种可能性也是通过适宜的标记/标签(例如，其偶联到基因产物的标记/标签)或者通过报告基因标记一种或多种代偿性上调的基因，其中该报告基因处于该代偿性上调基因的增强子和/或启动子的控制下，并且被导入生物。适宜的报告基因是技术人员公知的，并且这里适宜的报告基因具体为任何类型的发光蛋白(例如，GFP、BFP，等等)或者能够产生可检测信号的其他报告基因(例如，萤光素酶，β-半乳糖苷酶)和营养缺陷型株系的生长标记，例如HIS3、URA3、LEU2、TRP1和抗生素抗性基因，例如卡那霉素或G418抗性基因。还可设想其他类型的显型。
显型后，可通过适宜的方法(例如，测量增殖速度、细胞计数或测定大小或形态等，以及与不发生异源表达的表型比较)便利地检查所述显型的成功。
根据实施本发明方法的优选形式，通过缺失、诱变或过表达至少一种代偿性调节的基因实施显型。
根据优选实施方案，通过减小/消除代偿性差异表达或通过标记至少一种代偿性差异调节的基因实施显型。
关于这一点，异源表达可导致该生物的至少一种内源基因的代偿性上调和下调，但是也可导致一种或多种基因被上调和一种或多种其他基因被下调。
如果蛋白质或蛋白质片段的异源表达是可诱导的，那么也是尤其便利的。适宜的系统是熟练技术人员公知的，适宜的实例是半乳糖-或铜-调节的启动子、Tet-On Tet-Off系统，等等。这可能包括可诱导地开启生物的外来或内源基因的表达(可诱导的敲入)或可诱导地或完全关闭该生物的内源基因的表达(可诱导的敲除)。为此，遗传修饰适当地包括导入能够诱导表达蛋白质或蛋白质片段的载体，优选具有乳糖(GAL/GAL10)或铜(CUP1)调节的启动子的载体、四环素可诱导的载体或者组织特异诱导的启动子，如hsp 16-2、unc-119、unc-54、mec-7、或秀丽线虫中的myo-3。
根据优选实施方案，生物是秀丽线虫、原核或真核细胞，尤其优选酵母细胞，优选酿酒酵母株系的酵母细胞。
优选借助于cDNA或寡核苷酸微阵列，通过DNA/RNA分布图分析修饰的基因表达，但是该分析原则上包括mRNA或蛋白质稳定态(转录、翻译、稳定化，等)的任何修饰，因此也可通过蛋白质分布图并借助于蛋白质阵列实施。
在该方法的优选设计中，通过减小或消除代偿性差异调节实施显型。如果代偿性差异调节的基因的表达比对照生物中的更强，那么通过完全或部分抑制增强的表达实施所述减小或消除。这优选通过如下方法实施：与缺失株系杂交并随后选择双突变体(当生物为酵母时尤其适宜)，用适宜的载体进行基因组敲除(这技术人员所公知，并且同样非常适合于酵母，这里特别为酿酒酵母)，通过放射和/或诱变性物质的诱变或者导入反义载体或者抑制所述基因的蛋白质产生的类似载体。为此，尤其优选敲除的代偿性差异调节的基因，包括敲入报告基因，例如，β-半乳糖苷酶、萤光素酶或生长标记(如HIS3、ADE2、URA3)或者抗性标记，如卡那霉素抗性基因。然后报告基因可用作随后试验中检测和定量所试验药物的效能的信号。这包括优选用报告基因(例如，萤光素酶、β-半乳糖苷酶，等等)的编码序列(也包括所述序列足够被检测的部分)取代差异调节基因的至少部分编码序列。如果代偿性差异调节的基因的表达强度不如对照生物中的强，那么通过增强表达，优选通过杂交，导入附加体或者能够选择的另一表达载体或通过基因组敲入(上面的方法尤其适于使用酵母作为生物)实现减小或消除。优选减小或消除代偿性差异调节导致遗传修饰生物的生长抑制，但是其他表型也是有利的。
本发明的另一方面涉及通过本发明方法产生的遗传修饰的、显型的生物。
具体地，本发明涉及经遗传修饰生物，其具有至少一种内源或外来基因的遗传修饰的表达，该基因的表达导致所述生物的至少一种其他内源基因的代偿性差异调节，并优选因此终止或抑制可评估的/可检测的/有用的表型的出现，并且该生物具有于由减小/消除基因的代偿性差异表达或者标记代偿性差异调节的基因产物导致的表型。
本发明的另一方面涉及根据本发明制备的经遗传修饰生物在筛选对异源蛋白或蛋白片段的功能有影响的物质中的用途，还涉及鉴定对异源蛋白或蛋白片段的功能有影响的物质的方法。
根据另一方面，本发明还涉及使用本发明的显型生物筛选药物的测定法，其步骤是确定表型(例如，由于诱导的蛋白质异源过表达导致的生长抑制)，将所要测试的物质与所述生物接触，并观察所述表型的可能的修饰，优选至少部分转向野生型生物的行为或形态(即，至少部分恢复起始生物的表型，例如，结束生长抑制)。此外，还涉及通过本发明的方法或本发明的测定法鉴定为有效的物质。
下面基于实施例更详细地阐明本发明。
实施例1：基于以酵母为生物开发鉴定对激酶的活性起作用的药物的平台技术
在该情况中产生的表型是酵母的生长抑制。从而测定原理是基于酵母的生长抑制，它被用作活的“试剂管”。这里生长抑制指，例如，相关细胞的细胞周期抑制或裂解。使用酵母，是由于它们的可遗传操作性，从而使它们非常合适。人(或其他外源)激酶在酵母中过表达，并且处于半乳糖诱导的启动子(GAL1/10)的控制下。根据标准方法转化和培养酵母。所用的载体的实例是p41x-GAL1或p42x-GAL11系列的载体。
所有被测激酶中，约有30％过表达将已经导致酵母生长抑制(Tugendreich等人(2001))。该方法在图1中通过步骤1、3、5证明。那些过表达导致生长抑制的激酶被整合到适宜的酵母株系，然后转移到高通量筛选(HTS)。该实施例使用株系背景“MAT
a his3□1 leu2□0 met15□0ura3□0”的酵母株系(BY4741，来自EUROSCARF)。
在开发用于HTS的测定法中，通过测定上面描述的株系背景中的各种“药物转运蛋白”缺失突变体而优化条件。对于将要在该实施例中测试的所有蛋白质激酶，测定具有下面的缺失组合的株系：1.YRWS21(MAT
apdr1□::KanMX pdr3□::KanMX his3□1 leu2□0 met15□0 lys2□0ura3□0)2.YRWS39(MAT
a pdr5□::KanMX yor1□::KanMX his3□1leu2□0 MET15 lys2□0 ura3□0)3.YRWS14(MAT
a pdr5□::KanMXsnq2□::kanMX his3□1 leu2□0 MET15 lys2□0 ura3□0)4.YRWS13(MATa snq2□::kanMX yor1□::KanMX his3□1 leu2□0 MET15 lys2□0ura3□0)5.YRWS44(MAT
a pdr5□::KanMX snq2□::KanMX yor1□::KanMX his3□1 leu2□0 met15□0 lys2□0 ura3□0)。
然后可能在高通量筛选中搜寻减小或消除生长抑制，即导致酵母培养物的生长抑制的生物和化学分子。所有先前描述的技术对于熟练技术人员都是公知的。
如上面所描述的，所有外源激酶的约30％导致酵母生长抑制。因此，约70％所有过表达的激酶仅导致低的(如果存在)生长抑制。为了利用酵母的生长抑制原理作为所有蛋白质激酶的化合物筛选的平台技术，所剩余的70％蛋白质激酶必须也导致生长抑制。为此，需要本发明。
将所需的蛋白质激酶克隆到所选的酵母表达载体中，在该实施例中载体为p413 GAL1(D.Mumberg等人(1994)，全长并且具有C-末端标签，例如MYC标签)。用根据标准方案(见《酵母遗传学方法》(methods in YeastGenetice))的乙酸锂方法转化，并培养于适宜的培养基中后，通过根据标准方案加入半乳糖(20g/ml培养基)，在30℃下4到6小时诱导酵母中外源激酶的过表达。通过根据标准方案的免疫印迹，借助于针对所选标签的抗体(例如，抗-MYC：AB1364(Chemikon)或M5546(Sigma)；抗-HA：HA-11-A(Biotrend)或55138(ICN))检查激酶的表达。
免疫检测酵母中表达后，借助于DNA微阵列研究酵母中外源激酶的表达导致的基因表达的修饰(代偿性差异调节)。DNA微阵列是支持体材料，特定寡核苷酸被化学偶联到该材料。DNA微阵列被用作可覆盖完整酵母基因组的当前表达模式的工具。对于该类型实验，将激酶转化的酵母与作为对照的假转化的(空质粒)酵母比较。通过标准方法从两种株系制备总RNA。然后将RNA与芯片偶联的寡核苷酸(在微阵列上)45℃下杂交16h。激酶转化的酵母RNA与假转化的酵母RNA的直接比较揭示被过表达的蛋白质激酶以代偿性差别方式调节的酵母基因。本发明人的研究已经表明在例如外源蛋白质激酶的过表达中的基因干涉上调特定数目的酵母基因的RNA并且下调特定数目(表1)。这以人激酶PAK1的实例实施。
表1：2种基因被上调，11种基因被下调。
此外，本发明人能够首次证明许多被上调的基因由于代偿性原因而被上调。在该情况中，将酿酒酵母野生型株系(W303-1a(株系背景或供应源))与缺失酿酒酵母cla4(□cla4)基因的株系(YEL252)比较。除了缺失CLA4的基因，两种株系都是等基因的，即相同的。当直接比较来自两个不同株系(W303-1a和YEL252)的RNA制剂时，发现该酵母基因组的110种不同RNA被上调(表2)。
表2：56种基因被下调(数据未显示)。这里，特定基因的RNA拷贝数的增加可能是由于代偿性原因。在该具体实例中，代偿性指CLA4基因的缺失导致的遗传修饰株系中的缺陷可通过可以完全或部分接管CLA4功能的基因的表达增加而被减弱。为了证明该论点，选择一些被上调的基因用于进一步实验(见表3中的“第二个缺失”)。
表3：为此，选择MAT□酵母株系(其可以从，例如EUROSCARF或Research Genetics得到)，该株系在每种情况下缺失被上调的基因。该缺失由标记基因标记，即，例如抗生素抗性或者所需的生长因子的基因，例如，将特定氨基酸的基因整合到特定酵母基因组中。根据酵母遗传学的标准方法(Methods in Yeast Genetics：A Cold Spring Harbor CourseManual(1994))将所选的缺失株系与CLA4缺失株系(YEL252，MATa)杂交。
杂交后，选择二倍体酵母并诱导其形成孢子。这包括从二倍体酵母细胞产生4种单倍体孢子，这些孢子可以分成用于出芽的4种单倍体酵母克隆。因此，二倍体株系的基因被重新分配。在25％的所有情况中，不同起始株系的两种缺失将在一个新的单倍体克隆中联合。这可以基于各种选择标记容易地监控。
该标准方法被用于尝试制备13种不同的双缺失。在仅10例中，双缺失是可存活的，在3例中，双缺失从未发生(表3“致死的”)。在所有3例中，测试了40个子囊。因此明确两种缺失的组合导致受影响的孢子死亡。它们也是综合的致死的。已证明在所有13例中，双缺失或者是综合的致死的或者表现出其他综合的表型(表3)。该研究证明了这一论点，即为了代偿缺乏CLA4导致的缺陷，受影响的基因被上调。对本发明重要的是，在所研究的情况(13种双缺失)中，3种组合(因而是23％的所有可能的双缺失)显示出综合的致死性(表3)。
在使用□cla4株系的实验中，110种基因被上调(表2)。以同样的方式，上述方法中人PAK1的过表达上调了2种基因的mRNA(表1)。因此，这些基因也是由于代偿性原因被上调。由于被上调基因数目小而且基因上调与综合的致死组合连接的成功率低，所以我们在随后的实验中免除了鉴定在上调基因(具有表1的YMR096W或HIS3)中的缺失和人PAK1的表达的组合中显示出综合的致死表型的株系。相反，产生了人PAK1的超反应性突变体，即人PAK1□CRIB。再次使用标准方法将该突变体转化到酵母中。由于高激酶活性，该蛋白导致酵母生长抑制。已经鉴定了测定低分子量物质的适宜株系。已经实现了目标。然而，在该情况中，也使用DNA微阵列记录差异表达图谱，以便支持本发明的有效性(表4)。
表4：55种不同酵母基因被代偿性上调，由于高激酶活性，3种基因被下调(未显示)。如果PAK1突变体的的高活性不足够导致酵母中生长抑制，至此可能测定上调基因的缺失株系。PAK1突变株将不得不在特定缺失株系中表达。基于在综合的表型中23％的成功机会值，人PAK1突变株的表达将导致约13种酵母株系的生长抑制。从而，已鉴定用于测定潜在激酶抑制剂的株系。
对于测定酵母中人激酶的情况，起始株系将不需要被杂交，因为人激酶以依赖半乳糖的方式从质粒表达。所述质粒仅需要被转化到特定的缺失株系，并且该激酶的表达需要被诱导。在所要测定的23％的所有株系中，将可能观察到生长抑制(致死性)。由于特定缺失，生长被抑制的株系不再能够代偿质粒编码的蛋白质激酶的表达。因此，这些系统可被转移到HTS。
如果与DNA微阵列实验联合的特定野生型激酶的过表达不足以(如对于野生型PAK1所描述的，见表2)导致生长抑制，那么制备特定激酶的突变体并代替所述野生型激酶使用(同样对于基因表达实验，使用DNA微阵列)。可以根据随机诱变的原理制备这些突变株，目的是得到超活性突变体。对于诱变，根据Tugendreich等人(2001)的方法使用具有C-末端标签的激酶构建体。
从而，发明人的研究首次并且令人惊奇地表明缺失代偿性差异调节基因可导致生长抑制并且联系该发现设计了用于蛋白质激酶的标准平台测定法。在实际实验中，检测到生长抑制频率为23％。如上所述，通过优化(测试各种药物转运蛋白敲除)可以将用质粒编码的蛋白质激酶转化后显示出生长抑制的缺失株系转移到HTS。图1基于1、4、6-10点通过实例阐明了本发明。
除了杂交(cross-in)代偿性差异调节的基因，使用其他方法，如自身表达激酶的酵母的基因组敲除也可以实施基因缺失。然而，在酵母中，由于方法简单，通过杂交缺失或者基因组敲除消除代偿性差异调节的基因尤其有利。相比，其他方法可能更适于其他生物。从而，在例如真核细胞系和对于多细胞生物(如果蝇和秀丽线虫)，应用反义方法(如RNAi)更适宜。在每种情况中选择适于单个生物的测量方法在技术人员的能力范围内。
本发明的平台测定使得所有蛋白质激酶(如基于人PAK1所描述的)的HTS类似，从而是划算的测定系统。该系统也适于在化合物筛选中测定IC50值。
如实施例中描述的，基因表达实验还导致基因RNA的鉴定，这些基因受到外源激酶的表达的抑制。所述被抑制基因的启动子可用于HTS中的报告基因。为此，将酵母启动子与“报告基因”如β-半乳糖苷酶、萤光素酶、生长标记(如HIS3、URA3、LEU2或TRP1等)融合。然后将这些构建体转化到的酵母株系中用于HTS。在哪里它们可以作为消除受影响株系生长抑制的化合物的生长标记。
实施例2：该平台测定也可用作“多路系统”。多路系统指在一种反应混合物的同一测定中同时测定多种蛋白质或蛋白质片段，例如激酶。为此，首先构建单个显型的酵母株系。使用标准方法整合外源蛋白质激酶(见上文)。然后将这些酵母株系混合得到均一培养物。均一酵母株系混合物中的蛋白质激酶的表达导致生长抑制，因为每种单一激酶自身的表达导致显型酵母株系的生长抑制。HTS鉴定导致至少一种酵母株系的生长的化合物。然后必须确定与所述化合物相关的激酶。这通过“菌落PCR”方法(A.J.P.Brown和M.Tuite(1998))实现。为此，按照使用说明书(A.J.P.Brown和M.Tuite(1998))将几微升正生长的酵母培养物裂解。使用不同蛋白激酶的特定引物进行的定量RT-CPR从基因组DNA(的混合物)明确地鉴定被抑制的相关激酶(包括整合的蛋白质激酶)。从而，通过混合不同酵母株系的相等部分，可以在具不同激酶的单一筛选中测定。优点是节省大量成本和时间。
该技术不仅可应用于蛋白质激酶，而且可应用于导致酵母中转录应答的任一种蛋白质或物质。
该平台测定法在现有技术的测定对象中，使得例如所有蛋白质激酶(不仅那些其异源表达已经立即产生表型的激酶)的HTS类似，从而是划算的测定系统。该系统还适于在化合物筛选中测定IC50。
该技术不仅可应用于蛋白质激酶，而且可应用于导致酵母中转录应答的所有蛋白质或物质。
方法：
根据Sambrook等人(1989)《Molecular Cloning：A LaboratoryManual》，第二版，Cold Spring Harbor Laboratory Press，Cold SpringHarbor，NY.545页的标准方法用于基因操作。
根据Guthrie，C.和G.R Fink(1991)《Guide to Yeast Genetics andMolecular Biology》，卷194，J.N.Abelson和M.I.Simon编(San Diego，CA：Academic Press Inc.)对酵母(酿酒酵母)实施生长条件、杂交条件和基因操作。完全根据Klebl等人(2001)Biochem.Biophys.Res.Commun-286，714，720实施Affymetrix实验(“基因表达分析”)。
参考文献：
Brown，A.J.P.和M.Tuite(1998)，酿酒酵母中基于PCR的基因靶定.Methods Microbiol.26，67-81.
Methods in Yeast Genetics；A Cold Spring Harbor Course Manual；1994年版；Kaiser，C.，Michaelis，S.，和A Mitchell Cold Spring HarborLaboratory Press.
Mumberg，D.，Müller，R.和M.Funk(1994).酿酒酵母的可调节启动子：转录活性的比较和它们在异源表达中的用途.Nucl.Acids Res.22，5767-5768.
Tugendreich，S.，Perkins，E.，Couto，J.，Barthmaier，P.，Sun，D.，Tang，S.，Tulac，S.，Nguyen，A.，Yeh，E.，Mays，A.，Wallace，E，Lila，T，Shivak，D.，Prichard，M.，Andrejka，L.，Kim-R.和T.Melese(2001).使用基于酵母细胞的测定法平行地确定异源cDNA的表型的流水线方法。Genome Res.11，1899-1912.
表1：
表达hPAK1的ste20Δ株系YEL206中2种基因被上调
| 名称 | 基因功能 | 被上调的倍数 |
| YMR096W | 稳定期蛋白 | 2.15 |
| HIS3 | 咪唑甘油磷酸脱氢酶；组氨酸生物合成中的第七步 | 6.77 |
| 表达hPAK1的 | ste20Δ株系YEL206中11种基因被下调 | |
| 名称 | 基因功能 | 被下调的倍数 |
| STE20 | 信息素应答信号转导途径的丝氨酸/苏氨酸蛋白激酶 | 47.62 |
| FRE7 | 参与铁运输的与Fre1p和Fre2p稍微相似的蛋白质 | 11.70 |
| MFA1 | 交配信息素α-因子，通过Ste6p输出细胞 | 3.70 |
| YLR042C | 未知 | 3.27 |
| GPH1 | 糖原磷酸化酶，释放α-D-葡萄糖-1-磷酸 | 2.63 |
| FRE1 | 铁和铜还原酶，作用于Fe2+离子螯合剂 | 2.55 |
| YHR087W | 未知 | 2.31 |
| CWP1 | 细胞壁甘露糖肽素；PAU1家族的成员 | 2.27 |
| YJL217W | 未知 | 2.25 |
| CTR1 | 铜转运蛋白；铜离子的高亲和摄取所需要的； | 2.17 |
| FET4 | 低亲和铁(II)转运蛋白 | 2.00 |
表2：
cla4□株系YEL 252中110种基因被上调
| 名称 | 基因功能 | 被上调的倍数 | |
| 细胞壁维持 | FKS2 | □-1，3-葡聚糖合酶的组分，可能作为Fks1p的备选亚基(88％相同)；与Fks3p 55％相同，与Rho 1p相互作用，fks1Δfks2Δ是致死的。 | 6.81 |
| ECM29 | 可能与细胞壁结构或生物合成有关 | 3.13 | |
| SPI1 | 通过GP1锚结合到细胞壁；由Msn2/4p诱导 | 2.72 | |
| SBE22 | 芽生长所需，与细胞壁完整性有关 | 2.08 | |
| 细胞应急 | HSP12 | 12kDa热休克蛋白，由热、渗透(依赖HOG1-、PBS2的)或者氧化压力、稳定期、HSF1、MSN2、YAP1诱导；伴侣蛋白(hydrophilin家族成员)；5STREs | 6.55 |
| HSP26 | 热休克蛋白，由渗透压力、HSF1、MSN2、热、H2O2诱导；与Hsp42p 29％相同；伴侣蛋白；4STREs | 4.76 | |
| HSP82 | 热休克蛋白，与Hsc82p 97％相同，类似于哺乳动物HSP90(与人HSP90互补)；伴侣蛋白；由HSF1、SKN7、YAP1、H2O2诱导；具有ATP酶活性；被HOG1信号途径部分调节；结合Ste11p；HSP90活性受到Sch9p的调节 | 2.67 | |
| GPX2 | 谷胱甘肽过氧化物酶，由YAP1和氧化剂诱导 | 2.64 |
| SKN7 | 转录因子，参与氧化压力(H2O2)应答和G1细胞周期控制(出芽)；与Rho1p、Mbp1p、Cdc42p并且在基因上与PKC1相互作用；N2-撤退-诱导的假菌丝生长所需；在诱导基因表达中与Yap 1p协作；不参与热休克；可能参与HOG1信号途径；两组分系统的部分；依赖于Ras/PKA信号途径的skn7p刺激的转录活化。 | 2.60 | |
| SOD2 | 线粒体Mn2+超氧化物歧化酶，由HAP1、2、3、4、5诱导并且被cAMP(RAS2)抑制；对H2O2的转录应答依赖于Yap1p & Skn7p；由Msn2/4p诱导 | 2.57 | |
| ICT1 | 比野生型对Cu2+具有k.o.更高抗性；线粒体能量转移信号 | 2.41 | |
| CYP2 | Cyclophilin家族成员，热休克蛋白，异构酶，伴侣蛋白 | 2.37 | |
| HSP42 | 热休克蛋白，温和应急效应期间参与细胞骨架的恢复；由HOG1、MSN2/4、EtOH、H2O2诱导；3STREs | 2.28 | |
| MSN4 | 与Msn2p极相似；应急时调节海藻糖浓度；39种基因依赖于Msn2/4p诱导双峰偏移并且被cAMP抑制；ALD3、GDH3、GLK1、HOR2、HSP104、HXK1、PGM2、SOD2、SSA3、SSA4、TKL2、TPS1、ARA，例如Ras2p通过Msn2/4p& Yap1p控制应急应答基因表达；TOR信号转导控制营养物调节的转录因子的核定位。 | 2.15 | |
| 核苷酸代谢 | ADE2 | 磷酸核糖基氨基咪唑羧化酶(AIR去羧化酶)；白对红菌落 | 5.96 |
| ADE17 | 5-氨基咪唑-4-羧酰胺核糖核苷酸(AICAR)转甲酰酶/IMP环化水解酶；白对红菌落 | 3.42 | |
| DCD1 | Deoxycyticy1ate脱酰氨酶；k.o.具有增加的dCTP库 | 2.50 | |
| 小分子转运 | FRE7 | 参与铜和铁的摄取，与Fre1p弱相似性 | 4.98 |
| YHR048W | 与Ygr138p、Ypr156p 29％相同，与Flr1p 33％相同；MFS-MDR成员 | 4.20 | |
| PH089 | 高亲和Na+-依赖的磷酸转运蛋白 | 2.76 | |
| YGR138C | MFS-MDR的簇1(家族1)的成员，与Ypr156p89％相同 | 2.54 | |
| YER053C | MCF成员 | 2.40 | |
| TAF1 | 三乙酰基fuscerinine C转运蛋白(MDR-MFS)；与Arn1p、Ycl073p、Ykr106p分别56％、46％、46％相同 | 2.24 | |
| MUP3 | 低亲和力氨基酸通透酶(Met通透酶)；APC家族成员 | 2.16 | |
| ATM1 | ABC超家族成员，生长所需；可能在感知铁中起作用；与人ABC743％相同 | 2.03 | |
| 糖代谢 | GRE3 | NADPH-特异的醛糖还原酶，通过渗透压力、MSN2/4、0.1M LiCl诱导；与Yjr096p、Gcy1p、Ypr1p分别36％、34％和34％相同；STREs和PDSEs；类似于人305B蛋白(新生儿胆汁郁积性肝炎) | 3.61 |
| GPH1 | cAMP抑制的糖原磷酸化酶；应急诱导的 | 3.49 | |
| GUT1 | 甘油激酶，催化甘油向甘油-3-磷酸的转化，由ADR1、INO2、INO4、甘油诱导；与人GK非常相似；处于渗透压力时活性降低 | 3.37 |
| PCY1 | 丙酮酸羧化酶I；将丙酮酸转化成用于糖原异生的草酰乙酸；与Pyc2p、Hfa1p、Dur1，2p分别93％、30％、38％相同；类似于人PYC | 2.50 | |
| TSL1 | 海藻糖-6-磷酸合酶/磷酸酶复合体的组分；由STE12、STE7、TEC1、渗透压力诱导并且被cAMP、葡萄糖抑制；含有STREs | 2.40 | |
| GLK1 | 葡糖激酶特异的dor己醛糖；与Ydr516p、HxkIp、Hxx2p分别73％、38％、37％相同；由GCR1、HOGI、MSN2、MSN4诱导并且被cAMP、冷抑制；遇H2O2、Gl期时蛋白质增加。 | 2.09 | |
| 蛋白质降解 | YPS3 | 胞质膜上GPI-锚定的天冬氨酰蛋白酶(yapsin)；与Mkc7p、Sst1p、Yps1p分别45％、36％、47％相同 | 3.40 |
| UBI4 | 泛素聚蛋白，成熟泛素从聚泛素(Ubi4p)或者从核糖体蛋白Rps31p、RpI40Ap、RpI40Bp上切除；核糖体热休克蛋白&蛋白质缀合因子；与RpI40A/Bp 90％相同，与Rps31p 100％相同；HSF1、MSN2、饥饿、热休克诱导；细胞应急存活所需；k.o.对H2O2、N2-和C2-饥饿超敏感；有STREs和HSEs | 3.27 | |
| VID24 | 液泡输出和Fbp1p降解所需 | 2.82 | |
| RPN10 | 26S蛋白酶体复合体的非-ATP酶组分，体外结合泛素-溶菌酶缀合物；C-末端结合泛素 | 2.46 | |
| BUL1 | 参与泛素化途径，结合泛素连接酶 | 2.12 |
| AAP1 | Ala/Arg氨肽酶，与其他Zn2+金属蛋白酶&哺乳动物Zn2+氨肽酶有关 | 2.00 | |
| DNA合成 | RIM1 | 结合ssDNA的转录因子；线粒体中复制所需 | 3.27 |
| 氨基酸代谢 | YMR250W | 类似于谷氨酸脱羧酶 | 3.11 |
| GDH2 | 谷氨酸DH，从谷氨酸产生NH4 +的初级途径，由雷帕霉素诱导；应答N2饥饿而被磷酸化(失活；依赖PAK的) | 2.83 | |
| GCV1 | 甘氨酸脱羧酶T亚基，在Gly降解途径中起作用 | 2.31 | |
| CHA1 | 线粒体L-Ser/L-Thr脱酰氨酶，催化Ser向丙酮酸和Thr向□-丁酮酸的转化，由Ser、Thr、SIL1、CHA4诱导。 | 2.17 | |
| 信号转导 | YGL179C | Ser/Thr蛋白激酶，与Elm1p(31％)、Pak1p(49％)、Kin82p(30％)、Gin4p(29％)具有相似性 | 3.10 |
| KSP1 | Ser/Thr蛋白激酶，当过表达时抑制prp20Δ | 2.85 | |
| SLT2 | MAP激酶家族的Ser/Thr蛋白激酶，参与细胞壁完整化途径、极性生长、对营养可用性的应答、热休克；与Rlm1p、Swi4/6p、Mkk1/2p、Spa2p、Ptp2/3p、磷酸化Swi4/6p相互作用并且作为SBF复合体的调节剂；信息素诱导的激酶活性(需要Ste20p，但不需要Ste12p)；激酶活性受到细胞周期调节 | 2.77 | |
| STE20 | 信息素应答途径的Ser/Thr蛋白，还参与丝状生长和STE营养生长途径； | 2.25 |
| YCK1 | CKI同种型，与Yck2p、Yck3p、Hrr25p77％、50％、41％相同并且与人同种型50-55％相同；香叶基香叶基化(geranylgeranylated)；yck1Δyckts显示出超极性生长，对Zn2+和多种药物超敏性，Mn2+抗性 | 2.21 | |
| YHR046C | 肌-肌醇-1(或-4)-单磷酸酶，参与Ca2+信号的肌醇周期&肌醇生物合成；类似于人MYOP(抗躁狂和Li+的抑郁作用) | 2.17 | |
| SCH9 | 被cAMP激活的Ser/Thr蛋白质激酶；与Ypk2p、Ypk1p、Tpk3和人AKT1，2分别46％、44％、42％和49％相同；控制FGM途径；k.o.在假菌丝生长中具有适度缺陷并且显示出超侵染性生长 | 2.17 | |
| PTP2 | PTP酶，参与Hog 1p和信息素途径；与Hog1p、Slt2p相互作用；被SLT2、YAP1、热、渗透压诱导；使Hog1p、Fus3p去磷酸化，受到Hog1p的翻译后调节；2STREs | 2.01 | |
| 脂质、脂肪酸和固醇代谢 | PLB3 | 磷脂酶B，将GPI释放到培养基中 | 3.01 |
| ERG7 | 羊毛甾醇合酶(麦角固醇生物合成)，基本的 | 2.30 | |
| 膜融合 | YHR138C | 参与液泡融合，与Pbi2p具有序列相似性 | 2.81 |
| 细胞周期控制 | PCL5 | 与Pho85p结合的细胞周期蛋白，属于pcl1/2p亚家族 | 2.73 |
| PoIII转录 | GAT2 | GATA Zn2+指转录因子，N2分解代谢物阻遏敏感基因的表达所需的 | 2.73 |
| HAP4 | 转录因子，参与含有CCAAT盒基因(例如，SOD2)的活化的Hap2/3/4/5p复合体组分 | 2.48 | |
| STP4 | 转录因子，与Stp1，2，3p具有强同源性；参与tRNA剪接和支链氨基酸摄入 | 2.17 | |
| SNF6 | 转录因子，SWI-SNF全局转录活化复合体的组分；与SWI-SNF复合体直接相互作用的Gcn4p，Swi5p，Hap4p的酸性结构域 | 2.13 | |
| SET1 | 含SET-结构域的蛋白质trithorax家族的转录因子，参与转录和染色体结构的控制；类似于人HRX Zn2+指蛋白。 | 2.04 | |
| 能量产生 | MDH2 | 胞质苹果酸DH(乙醛酸循环)；N2源缺陷诱导并且被cAMP、葡萄糖抑制；3STREs | 2.60 |
| RNA加工/修饰 | RPP1 | 核糖核酸酶P和RNA酶MRP核糖核蛋白颗粒的亚单位，tRNA和5.8S rRNA加工所需；与hRpp30 23％相同 | 2.49 |
| PRP8 | U5snRNA-相关的剪接因子；基本的RNA-结合蛋白；与人PRP862％相同；剪接体的组分 | 2.41 | |
| RRP4 | 核糖体5.8S rRNA的3’加工所需的3’-5’-外切核糖核酸酶；3’-5’-外来体复合物的核和胞质形式的组分；基本的；在S-期诱导 | 2.38 | |
| DBP8 | 类似于RNA解旋酶的DEAD盒家族 | 2.33 | |
| 其他代谢 | YNL274C | 潜在的□-酮异己酸还原酶，被YAP1，H2O2诱导 | 2.26 |
| DUR1，2 | 尿素酰胺分解酶(Urea amidolyase)，含有尿素caroxylase和脲基甲酸酯水解酶活性；被NH4 +抑制并且被N2饥饿、交配信息素、Arg、雷帕霉素(rapamycin)诱导(N2利用基因) | 2.21 | |
| 蛋白质修饰 | UBP5 | 泛素-特异的蛋白酶，与Doa4p和人Tre-2同源；硫氰酸酶同源家族成员 | 2.17 |
| 蛋白质合成 | MSR1 | 线粒体精氨酰-tRNA合成酶，与Ydr341p61％相同 | 2.17 |
| 囊泡转运 | SFB3 | COPII小泡的可能组分，与Pma1p从内质网向高尔基体的运输有关；与Sec23p相互作用 | 2.17 |
| 细胞因子 | CDC12 | 颈部septin复合体的基本部分；信息素诱导的形态发生所需要的；septin装配依赖于Cla4p & Ste20p(Cdc42p、Cdc24p)；在yck2ts中错误定位。 | 2.09 |
| 交配应答 | SSF1 | 不育four的抑制剂；与Ssf2p 94％相同；ssf1□ssf2□是致死的；hsp90损失功能突变的多拷贝抑制剂 | 2.06 |
| 未知 | YHR214W | 与Yar066p、Yil169p、Yol155p分别100％、77％、74％相同 | 9.88 |
| YAR066W | 与Yhr214p、Yil169p、Yol155p分别100％、77％、74％相同 | 7.59 | |
| RTA1 | 抗氨基胆固醇；由TEC1、STE7、STE12诱导。 | 4.64 | |
| MSC1 | 在减数分裂同源的染色单体重组途径中起作用 | 4.62 | |
| YHL021C | 由STE12、TEC1、STE7诱导 | 4.35 | |
| YHR209W | 推定的依赖SAM的甲基转移酶 | 4.26 | |
| Cos8 | 保守序列的蛋白质家族 | 3.74 |
| YNR014W | 与Ymr206p 30％相同；4个推定的STREs | 3.44 |
| YlR042C | - | 3.37 |
| YCL049C | - | 3.28 |
| YHR087W | - | 3.19 |
| YHR078W | 4个潜在的跨膜片段 | 3.00 |
| TRA1 | Ada-Spt转录调节复合体(SAGA)、类似SAGE复合体和NuA4复合体的基本组分 | 2.82 |
| BTN2 | 使用yhc3Δ升高表达；与人HOOK138％相同 | 2.77 |
| VAB36 | 36kDa Vac8p结合蛋白；2个推定的STREs | 2.75 |
| YFL063W | 与subtelomeric蛋白相似 | 2.68 |
| YHR112C | 与cystathione□-合酶Str2p和其他转硫酶相似，还与人CGL(胱硫醚尿)相似 | 2.56 |
| YBL064C | 1-Cys家族的线粒体硫醇过氧化物酶；S.c.中4种过氧化物酶之一；使用硫氧环蛋白作为电子供体；氧化压力时被诱导；存在DTT时还原H2O2。 | 2.55 |
| YSC83 | 孢子形成期间诱导的mRNA水平 | 2.46 |
| BOP1 | PAM1的旁路(PAM1＝PP2A损失的多拷贝抑制剂) | 2.45 |
| YHR045W | 5个可能的跨膜结构域 | 2.44 |
| YHR033W | 被N2源缺陷诱导并且被cAMP抑制 | 2.42 |
| YPR009W | 推定的Zn2+指结构域；与Sut1p 34％相同 | 2.40 |
| YLL064C | Seripauperin家族的成员 | 2.39 |
| YPL137C | 类似于Mhp1p(27％)、Yor227p(43％) | 2.39 |
| YHR182W | - | 2.37 |
| YDR222W | - | 2.37 |
| YHR146W | 类似于信息素适应蛋白Mdg1p | 2.36 |
| YMR184W | - | 2.36 |
| YGL261C | seripauperin(PAU)家族的成员 | 2.34 |
| YHR083W | 基本的 | 2.32 |
| YHR122W | 基本的 | 2.29 |
| YOR227W | 与Ypl137p、Mhp1p 43％、25％相同 | 2.27 |
| YHR186C | WD40结构域；基本的 | 2.26 |
| YHR073W | 类似于人氧固醇结合蛋白；与Spo12p相互作用 | 2.20 |
| YJL217W | - | 2.17 |
| YHR192W | - | 2.11 |
| YDL231C | 推定的Zn2+指结构域 | 2.10 |
| YDR391C | 与Yor013p、Yor012p 57％、41％相同 | 2.05 |
表3：
| 株系名称 | 第一个缺失 | 第二个缺失 | 表型 |
| W303-1a | - | - | 无 |
| EL252.1a | cla4 | - | 胞质分裂 |
| YAS | cla4 | ptp2 | 综合的 |
| YAS | cla4 | glk1 | 综合的 |
| YAS | cla4 | msn4 | 综合的 |
| YAS | cla4 | ygl173 | 综合的 |
| YAS | cla4 | gut1 | 综合的 |
| YAS | cla4 | rta1 | 治愈的 |
| YAS | cla4 | skn7 | 综合的 |
| YAS | cla4 | pde2 | 综合的 |
| YAS | cla4 | yck1 | 综合的，生长极慢 |
| YAS | cla4 | sbe22 | 综合的 |
| YAS | cla4 | elm1 | 致死的 |
| YAS | cla4 | slt2 | 致死的 |
| YAS | cla4 | ste20 | 致死的 |
表4：
在表达hPAK1ΔCRIB的ste20Δ株系YEL206中被上调的55种基因
| 名称 | 基因功能 | 被上调的倍数 |
| PHO5 | 可抑制的酸性磷酸酶；活化需要糖基化 | 10.19 |
| ZRT1 | 高亲和力锌转运蛋白；ZIP家族的成员 | 10.12 |
| PHO11 | 分泌的酸性磷酸酶 | 7.67 |
| HSP30 | 热休克蛋白，定位于质膜 | 6.30 |
| PHO12 | 分泌性酸性磷酸酶 | 5.80 |
| YIL057C | 未知 | 5.70 |
| YOL154W | 与锌金属蛋白酶具有相似性的蛋白 | 5.24 |
| YPL274W | 高-亲和性S-腺苷甲硫氨酸通透酶 | 5.16 |
| CIT3 | 线粒体柠檬酸合酶 | 5.15 |
| RTA1 | 参与7-氨基胆固醇抗性的蛋白 | 5.14 |
| YEL070W | 与大肠杆菌D-甘露糖酸氧化还原酶相似的蛋白 | 5.09 |
| YDL037C | 与葡聚糖1，4-□-葡糖苷酶相似的蛋白 | 4.95 |
| YHR136C | Pho80-Pho85p依赖细胞周期蛋白的激酶复合体的推定抑制剂 | 4.84 |
| LEE1 | 未知 | 4.59 |
| YMR303C | 乙醇脱氢酶II；将乙醇氧化成乙醛 | 4.07 |
Claims (22)
1.产生用于药物筛选的经遗传修饰生物的方法，该方法包括以下步骤：
a)通过对生物的遗传修饰导致异源表达至少一种蛋白质或蛋白质片段，
b)分析改变的基因表达模式并鉴定代偿性差异调节的基因，
c)显型该生物。
2.权利要求1所述的方法，其中通过减小/消除代偿性差异表达或者标记至少一种代偿性差异调节的基因实施显型。
3.权利要求1或2所述的方法，其中遗传修饰导致至少一种蛋白质或蛋白质片段的异源表达，所述蛋白质或蛋白质片段是该生物内源的和/或外来的。
4.权利要求1到3中任一项所述的方法，其中遗传修饰导致至少一种该生物内源的蛋白质表达的减小或消除。
5.权利要求1到4中任一项所述的方法，其中改变的表达是可诱导的。
6.权利要求5所述的方法，其中遗传修饰包括导入一种载体，该载体使得该蛋白质或蛋白质片段可诱导地表达，优选用半乳糖、铜四环素(copper tetracycline)可诱导载体或其他相当的可诱导载体。
7.权利要求1到6中任一项所述的方法，其中遗传修饰包括敲除，优选可诱导的敲除。
8.权利要求1到7中任一项所述的方法，其中生物是果蝇、秀丽线虫、原核或真核细胞。
9.权利要求8所述的方法，其中细胞是酵母细胞，优选酿酒酵母株系的酵母细胞。
10.权利要求1到9中任一项所述的方法，其中借助于DNA微阵列或蛋白质微阵列分析改变的基因表达。
11.权利要求1到10中任一项所述的方法，其中通过减小或消除代偿性差异调节的基因的表达实施显型。
12.权利要求11所述的方法，其中与对照生物相比，代偿性差异表达的基因的表达有所增强，并且通过至少部分抑制所述增强的表达导致减小或消除。
13.权利要求7所述的方法，其中通过用报告基因的编码序列或者通过用足以被检测的部分报告基因序列替换差异调节的基因的至少部分编码序列，实施差异表达基因的敲除。
14.权利要求11所述的方法，其中差异表达的基因不如对照生物中的表达强，并且通过增强其表达实施减小或消除。
15.权利要求1到14中任一项所述的方法，其中减小或消除导致生物的生长抑制。
16.权利要求1到10中任一项所述的方法，其中通过标记代偿性差异调节基因的基因产物实施显型。
17.通过权利要求1到16中任一项所述的方法得到的经遗传修饰的显型生物。
18.经遗传修饰生物，其具有
a)至少一种内源或外来基因的经遗传修饰的表达，该表达导致所述生物的至少一种其他内源基因的代偿性差异表达，和
b)通过减小/消除基因的代偿性差异表达或者通过标记该代偿性差异调节的基因产物导致的表型。
19.权利要求17或18所述的经遗传修饰生物的用途，用于筛选对异源蛋白质或蛋白质片段的功能有影响的物质。
20.鉴定对异源表达的蛋白质或蛋白质片段的功能有影响的物质的方法，该方法包括使用权利要求17或18所述的生物。
21.使用权利要求17或18所述的至少一种表型生物筛选药物的测定法，该测定法包括步骤
a)确定所述生物的表型；
b)将受试物质与所述生物接触；
c)观察所述表型的可能的改变。
22.一种物质，其被如权利要求20所述的方法或者如权利要求21所述的测定法鉴定为至少减小表型的物质。
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| JP2009515997A (ja) * | 2005-11-18 | 2009-04-16 | タケダ サン ディエゴ インコーポレイテッド | グルコキナーゼ活性剤 |
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| AU2008245696B2 (en) | 2007-04-27 | 2013-11-07 | Pelican Technology Holdings, Inc. | Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins |
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- 2003-11-18 KR KR1020057011453A patent/KR101215854B1/ko not_active IP Right Cessation
- 2003-12-16 US US10/736,801 patent/US20040143854A1/en not_active Abandoned
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2005
- 2005-05-24 ZA ZA200504212A patent/ZA200504212B/en unknown
- 2005-07-08 NO NO20053344A patent/NO20053344D0/no not_active Application Discontinuation
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110172454A (zh) * | 2019-05-23 | 2019-08-27 | 浙江大学 | 一种s-腺苷甲硫氨酸合成酶突变体及其高通量筛选方法 |
| CN110172454B (zh) * | 2019-05-23 | 2020-11-13 | 浙江大学 | 一种s-腺苷甲硫氨酸合成酶突变体及其高通量筛选方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2003283409A1 (en) | 2004-07-09 |
| KR101215854B1 (ko) | 2012-12-31 |
| HK1087734A1 (en) | 2006-10-20 |
| NO20053344L (no) | 2005-07-08 |
| WO2004055206A1 (de) | 2004-07-01 |
| JP2006509512A (ja) | 2006-03-23 |
| NO20053344D0 (no) | 2005-07-08 |
| ATE508201T1 (de) | 2011-05-15 |
| ZA200504212B (en) | 2006-02-22 |
| US20040143854A1 (en) | 2004-07-22 |
| KR20050085806A (ko) | 2005-08-29 |
| EP1576186B1 (de) | 2011-05-04 |
| DE10258885A1 (de) | 2004-07-15 |
| KR20120038025A (ko) | 2012-04-20 |
| CA2509333A1 (en) | 2004-07-01 |
| DE50313671D1 (de) | 2011-06-16 |
| RU2005122470A (ru) | 2006-05-10 |
| JP4630067B2 (ja) | 2011-02-09 |
| RU2376364C2 (ru) | 2009-12-20 |
| EP1576186A1 (de) | 2005-09-21 |
| CN1726290B (zh) | 2012-09-05 |
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