Tools and Technologies I
With the complete sequencing of the human genome, the current challenge is to learn how the products of the 30,000 – 150,000 identified genes interact to produce the complexed human body, which contains very diverse cell types with diverse functions, and how diseases arise when either the functions of these genes or their regulations go awry. To meet this challenge, we need to know what genes are being expressed at a particular time in a particular cell. Currently technology is being developed to study the expression of many genes at once. However these technologies are limited to study gene expression in millions, or thousands of cells, thereby restricting the study of small samples or complex tissues, especially cells that are very rare in the body, such as the stem cells. To understand how genes regulate stem cells maintaining their “stemness” and differentiation, we propose a strategy to study gene expression in individual cells by sequencing all the transcripts in a single neural stem cell taken out from a developing mouse embryo, by combining two latest technologies – Connector Inversion Probe (CIP) technology and the SOLiD Sequencing platform from Applied Biosystems, which is one of the latest DNA sequencing technology that has a capacity of 114 million sequencing reactions per run. The CIP technology allows the rapid cloning and amplification of all the transcripts in the cell. In the current CIRM proposal, we propose to further develop the CIP technology, and to test our strategy to sequence all the transcripts in a single cell, we propose to sequence 1000 genes in individual neural stem cells. If this strategy proves successful, it will provide deep understanding how many human diseases arise at a single cell level, and facilitate scientists and physicians to develop novel and effective therapeutic treatments.
Statement of Benefit to California:
Almost all human illness, perhaps except trauma, have some genetic basis. While the complete human genome is sequenced, it is still not clear how mutation in gene function or alteration in their regulation leads to human diseases. In this CIRM proposal, we propose to develop a strategy to sequence all the transcript in a single cell. If this strategy is successful, it will enable scienctists and physicians to have a deep understanding how certain human diseases arise and progress at a single cell level. It will guide the development of novel diagnosis and/or treatment strategies. Everyone, include the citizens of California, will benefit tremendously from this research.
This application focuses on the development of technology for large-scale, high-throughput mRNA sequencing of individual neural stem cells (NSCs) from cerebral cortex. The applicant proposes to use two recently developed technologies that are commercially available: Connector Inversion Probes (CIP) and the SOLid sequencing platform. The CIP technology permits amplification of selected mRNAs from individual, identified NSCs within populations of tens or hundreds of cells. Subsequent high-throughput sequencing via SOLid would produce transcriptional profiles of these NSCs, which could be used to investigate transcriptional heterogeneity within a population and its effects on neural differentiation. The applicant plans to first optimize probe-generation methods for single cell gene expression before scaling up to larger numbers of cells. After scale-up and optimization of high throughput sequencing, the applicant proposes to develop new software tools for automated probe design and sequence analysis. The reviewers were enthusiastic about the technology in this proposal but felt that the case was poorly made for its application to stem cell biology. The proposal is complex and lacking details, and reviewers expressed concern about the biological and clinical relevance of the work. Finally, reviewers noted a lack of staff dedicated to the project. The impact that this technology would have on the field was not clear. Reviewers agreed that the identification of specialized probes containing molecular bar codes for transcript profiling of individual stem cells from the cerebral cortex would be of high impact. However, quantification of specific mRNAs can already be done by a number of technologies, and the applicant did not clearly explain the added value of the chosen technology. One reviewer commented that sequencing each mRNA could be important because it could lead to the identification of previously undetermined splice patterns, but this reviewer was disappointed that the applicant did not propose to investigate alternative splicing in NSCs as a mechanism for generating cell diversity. Reviewers raised concerns that the applicant proposed technology development for its own sake, rather than with an eye toward advancing the field of stem cell biology. In general, the reviewers expressed doubts about the project’s biological and clinical relevance and felt that its potential impact on stem cell biology could have been developed in much greater detail. In terms of feasibility, while reviewers agreed that the investigator and listed collaborator have the expertise to accomplish the work described, they were concerned that the application lacked experimental details. One reviewer noted a dearth of cell biology in the application, including details such as how NSCs would be isolated and selected. For instance, it is unclear whether the cells subjected to CIP would be cultured or acutely dissociated from the brain. Methods for single cell amplification of mRNA were also sparse. In spite of the lack of detail, reviewers commented that the experimental plan seemed robust and felt that it was appropriate to validate the technology before proceeding to the high throughput methods. Feasibility of the technology has been demonstrated by earlier applications of CIP. Finally, reviewers appreciated the discussion of milestones and pitfalls, especially given the complexity of the proposal. The applicant and the proposed collaborator were described as qualified to conduct the experiments described. The PI has strong background in developmental neurobiology while the collaborator has well documented expertise with the CIP technology. In terms of the budget, one reviewer noted that, besides partial salary support for the PIs, the proposal would only fund one full-time graduate student and one half-time technician and wondered if this staffing would be sufficient to complete the project. Several other issues were raised with the budget, including its high composition of consumables and the high cost of sequencing. One reviewer commented that the requests for some items of equipment were not adequately justified in the application. Overall, while the technologies described in this proposal are powerful and promising, the applicants did not make a strong case for their potential to advance the field of stem cell biology.