Tools and Technologies I
The ability of cells to continue to divide in prolonged culture allows researchers an unprecedented opportunity to introduce genes into the genome and select clones with the appropriate genetic modification. The development of a variety of vectors and strategies has allowed perhaps the most dramatic examples of this in non-human stem cell populations – the use of mouse embryonic stem cells (mESCs) to obtain truly remarkable modifications of the mouse genome. In principle, these various strategies could be adapted to human stem cells. The human stem cell field gained momentum when human embryonic stem cells (hESCs) were successfully isolated in 19981. hESCs present an unparalleled opportunity to fully benefit from the advantages of genetic manipulations in a controlled manner, in particular, gene targeting via homologous recombination. In theory, by developing techniques for gene targeting in hESCs, it is possible to analyze the function of any gene and to develop in vitro models for developmental studies or for the purification of cells of specific somatic lineages. However, the techniques that have been successfully used in targeting mESCs have met unexpected challenges when adapted to hESCs, and to date, only three genes have been targeted in hESCs. One major difficulty in targeting hESCs is the complex DNA constructions required for homologous recombination. We believe that elaborate targeting vector construction is a prerequisite for successful gene targeting in hESCs. To generate such targeting vectors rapidly, more efficient cloning techniques are required. For example, instead of the traditional strategy of restriction digestion and ligation, which is often laborious and ineffective for cloning large DNA fragments required for homologous recombination, ET cloning approach via homologous recombination allows for direct isolation of human genomic DNA from BACs or yeast artificial chromosomes, thereby eliminating the step of prior isolation of large DNA fragments. In this proposal, we will develop novel targeting vectors utilizing ET cloning directly from human BACs for homologous recombination in hESCs. In particular, we will develop a GFP knock-in approach to target key genes involved in dopaminergic differentiation for which we have a well-defined system for characterization. We plan to target three transcription factors that are essential for specification and maturation of dopaminergic neurons in mice.
Statement of Benefit to California:
The ability to target a specific gene in mouse embryonic stem cells via homologous recombination has revolutionized the study of mammalian gene function. Our proposed research will develop novel tool/technique for targeting human embryonic stem cells via homologous recombination. In particular, we will target genes that are know to play an important role in the development of dopaminergic neurons, neurons that are selectively and progressively degenerated in patients with Parkinson’s disease. Such targeted cells will be very useful for studying the roles of these genes in dopaminergic differentiation of hESCs which may provide insights into pathology and treatment of Parkinson’s disease as well as new therapy development. Therefore, the proposed research will also benefit California residents suffering from PD.
This proposal focuses on the development of novel targeting vectors for efficient homologous recombination (HR) in human embryonic stem cells (hESCs) utilizing novel cloning technologies. The applicants propose to use bacterial artificial chromosome (BAC) based cloning and HR techniques to improve the efficiency of gene targeting in hESCs. Specifically they plan to create hESC reporter lines with GFP inserted into the pro-dopaminergic transcription factors Lmx1a, Nurr1 and Pitx3. They then propose to demonstrate the utility of these knock-in reporter lines by selecting dopaminergic neurons from hESC cultures by fluorescence activated cell sorting (FACS) and functional testing of these neurons in vivo in a rat model of Parkinson’s disease. Overall reviewers found the potential impact of this proposal to be modest. They believed the project to be feasible, although some concerns were expressed in terms of achieving the proposed milestones on time. In terms of the research team, reviewers found the technical expertise appropriate, although the publication track record was considered weak. Reviewers agreed that the stated goals of the proposal are important ones, however, they questioned the novelty and impact of the approach. They recognized the need for methods that improve the efficiency of HR in hESCs but noted that the use of longer recombination arms allowed by BACs is one of several reasonable approaches to optimization, and is not particularly novel. Reviewers appreciated the potential value of dopaminergic reporter hESC lines for Parkinson’s disease research. On the other hand, they commented that the three transcription factors mentioned in the proposal, Lmx1x, Nurr1 and Pitx3, likely contribute to fate determination for other cell types and therefore questioned the lack of inclusion of a marker of more terminally differentiated dopaminergic neurons, such as tyrosine hydroxylase. Finally, a reviewer noted that the applicants only propose to use one starting hESC line and felt that, given the variability in these lines, the impact would be greater if knock-ins were made in multiple lines. Reviewers agreed that the proposal is feasible mostly based on the preliminary data presented. One reviewer commented that the milestones are clearly described but may be difficult to reach in the two-year timeframe. The reviewer noted that the use of an ET cloning approach should help by eliminating an essential step of large DNA fragment isolation. Another reviewer was not sure that the chosen cell line, BG01, was a good choice. The karyotype of BG01 is not clearly stated in the preliminary data and the reviewer believes that this line has a propensity to develop an abnormal karyotype. Finally, a reviewer noted that it was unclear how the relative numbers of differentiated and undifferentiated cells would be determined following transplantation. Overall, reviewers found the research team to be qualified to carry out the proposed research. One reviewer would have liked to see a greater than 10% effort by the principal investigator, but noted that s/he is already is funded by 2 CIRM initiatives and other projects. Two reviewers commented that the requested budget of ~$500,000 per year seems excessive, particularly for a project with a linear workflow and downstream experiments dependent on successful gene targeting. Overall, while the reviewers felt this proposal was feasible and came from a strong research team, they questioned the impact and novelty of the approach.