罗氏454完成食蟹猴基因组测序并研发其表达谱芯片用于药物安全性研究

【字体: 时间:2011年11月08日 来源:罗氏诊断

编辑推荐:

  一个由来自罗氏制药早期研发部和罗氏子公司NimbleGen科研人员组成的研究小组,最近在Genome Research杂志上报道他们已经获得了第一稿的食蟹猴(Macaca fascicularis)的基因组序列草图,并开发了一个新的食蟹猴表达芯片设计,用于临床前药物安全研究。

一个由来自罗氏制药早期研发部和罗氏子公司NimbleGen科研人员组成的研究小组,最近在Genome Research杂志上报道他们已经获得了第一稿的食蟹猴(Macaca fascicularis)的基因组序列草图,并开发了一个新的食蟹猴表达芯片设计,用于临床前药物安全研究。

食蟹猕猴是基础医学和应用生物医学研究中最重要和最广泛使用的非人类灵长类动物模型之一。与其他物种相比,非人类灵长类动物与人类有更密切的进化关系,并具有较高的生理相似性,非常适合作为临床前药物安全性评估的模型。

要提高灵长类动物实验结果转化到人类应用的能力,罗氏分子毒理学全球负责人Ulrich Certa先生和他的团队使用罗氏454公司的基因组测序FLX系统以及其他二代测序技术,应用shotgun法解码一个来自毛里求斯的雌性食蟹猴基因组,测序深度达到6倍基因组。此外,他们发现了约200万潜在的单核苷酸多态性(SNP),这将使得这个物种的高分辨率个体基因分型成为可能。

结合序列比对和外显子片段大小保守估计方法,对超过两万个食蟹猴基因组转录子进行了预测,并使用罗氏NimbleGen的12x135K芯片格式设计了一个食蟹猴特异性基因表达芯片,在不到两个月的时间实现了经济且全面的食蟹猴转录组分析。这种同时可以测试12个样本的芯片,每个样本有13万5000个长度为60个碱基的寡核苷酸探针,每个转录子通过6个探针进行检测。此外,芯片上还有剩余探针可以设计其他食蟹猴的基因组区域,或者其他客户定制的内容。

点击索取NimbleGen基因表达芯片的更多资料

研究人员对来自不同地区的36个食蟹猴肝脏样本的表达谱进行了比较分析,结果表明有超过700个表达高度可变,而大多数转录子在个体间的表达量相对稳定。有趣的是,许多药品安全及药物作用的相关基因的表达水平在实验中显示出存在相当的个体间以及种间差异。在个体之间的基因表达的差异对于分析药物安全至关重要。全基因组基因表达分析亦可以提高药物安全性研究的质量,并发现新药在相关的动物模型中的代谢及作用机理。

罗氏负责该项目的 Ulrich Certa先生说:“NimbleGen的表达微阵列平台的灵活性和准确性,使我们能够根据我们的基因组测序工作结果开发一种新基因芯片设计。食蟹猴的基因表达首次可以进行高特异性的监测,通过药物引起的基因转录水平的改变来解答药物作用机制或药物安全的问题。此外,我们计划设计监测拷贝数改变和SNP分析的NimbleGen芯片。这些方法相结合将有望提高非人类灵长类动物实验结果用于预测人类实验结果的可参考性。例如,在未来,在人类研究中同样可能发现基因多态性引起的对同一药物不同的代谢反应。”

罗氏非临床药物安全早期研发(pRED)的全球负责人Thomas  Singer说:“利用食蟹基因组以及NimbleGen基因芯片的表达谱分析数据,我们增强动物模型用于人类药物安全实验的作用与意义。尤其是,所发表的研究数据有助推动全球的实验动物的"3R"倡议,即对减少动物实验(reduce)、改善动物实验条件质量(refine)、替代动物实验(replace)有显著贡献。”

原文

Roche Researchers Sequence Complete Genome of Cynomolgus Monkey and Develop Novel Gene Expression Microarray for Drug Safety Assessment

Ateam of researchers from Roche including scientists from Roche Pharma Research and Early Development and Roche NimbleGen, reported inGenome Research (1) that they have generated the first draft genome sequence of the cynomolgus monkey (Macaca fascicularis) and developed a novel microarray design for in depth expression profiling for use in preclinical drug safety research.

The cynomolgus macaque is one of the most important and widely used non-human primate animal models in basic and applied biomedical research. Compared to other species, non-human primates have a closer evolutionary relationship to humans and exhibit high physiological similarity well suited to serve as translational models for preclinical drug safety assessment.

To improve the predictive power of primate experiments for humans, Prof. Ulrich Certa, Global Head Molecular Toxicology, and his team first applied a shotgun sequencing strategy using the Genome Sequencer FLX System from Roche’s 454 Life Sciences division in combination with other next generation sequencing technologies to decode the entire genome of a Macaca fascicularis female of Mauritian origin with 6-fold coverage. In addition, roughly two million potential single-nucleotide polymorphisms (SNPs) were discovered which, for the first time, will allow high-resolution genotyping of individuals in this species.

Using a combination of sequence alignment and exon size conservation, more than 20,000 transcripts in the cynomolgus monkey genome were predicted and used to build a M. fascicularis-specific gene expression microarray on the Roche NimbleGen 12x135K platform, in less than two months enabling comprehensive yet economical transcriptome analysis. The 12-plex expression array format contains 135,000 oligonucleotide features per array with six 60-mer probes interrogating each transcript with more probe space available for either including additional targets of M. fascicularis or other custom desired content.

The comparative expression analysis of liver samples from 36 animals of different geographic origin resulted in the identification of over 700 genes with highly variable expression while the majority of the transcriptome showed relatively stable expression with low inter-animal variation. Interestingly enough, considerable inter-individual as well as inter-species variability was found in gene expression levels of a number of drug safety and response related genes. Variation in gene expression among individuals can be critical for the interpretation of drug safety data and genome-wide gene expression profiling can now be used to improve drug safety studies and discover the mode of action of novel drugs in a relevant animal model.

“The flexibility and accuracy of the NimbleGen expression microarray platform allowed us to develop a novel microarray based on our genome sequencing effort. For the first time, gene expression can be monitored with high-specificity in this animal model to answer mechanistic or safety related questions based on transcriptional responses. Furthermore, we plan to design additional arrays for copy-number-variation and SNP analysis on the NimbleGen array platform. These combined efforts will hopefully improve the translational value of non-human primate experiments for humans. As in humans, it might become possible in the future to discover polymorphisms in drug-response genes that differentiate poor and good metabolizers for instance” explained Prof. Ulrich Certa, principle lead of the project.

“The knowledge we obtained from the cynomolgus genome and gene expression profiling using NimbleGen microarrays is an important contribution towards the better use of this species as a drug safety model for the assessment of novel human drugs. In particular, the published research data represents a significant contribution to the global “3R” animal welfare initiative, which has the goal to reduce, refine and replace animal experiments” stated Thomas Singer, Global Head of Non Clinical Safety Pharma Research and Early Development (pRED).

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