Tools and Technologies I
Gene targeting refers to the practice of introducing genetic modifications to cells with the goal to inactivate a specific gene or alter its biochemical properties. Over the last twenty years, this has been a major approach that investigators use to delineate in vivo function of genes in the mouse, the mammalian genetic model organism. Unfortunately, gene targeting technologies for human ES cells are still immature. Here we propose to develop and optimize the method for gene targeting in several widely used human ES cell lines. We will compare a conventional method routinely used for gene targeting in mouse ES cells and an recombinant Adeno-Associated Virus (rAAV)-based method that is increasingly being used for gene targeting in human somatic cell but not yet in human ES cells. We will first compare the time requirement, experimental complexity and efficiency of these two methods in knocking out one test gene in five human ES cell lines. We will experiment with various parameters in an attempt to optimize stable transfection and gene targeting frequency across different human ES cell lines. Once we identify the best protocol for gene targeting, we will apply the method to targeting of eight genes in human ES cells, to determine the robustness and generality of our gene targeting method. Successful completion of this project will lead to a robust and generally applicable gene targeting method for human ES cells. Such a tool will be critical for understanding the functional roles of various genes in ES cell proliferation and differentiation. It will also greatly facilitate the development of ES cell based therapeutics by, for example, enabling the repairing of faulty genes in patient specific ES cells or induced pluripotent stem (iPS) cells.
Statement of Benefit to California:
The overall aim of this application is to develop a robust and generally applicable gene targeting method for use in human ES cells. Development of such technology is highly significant if we are to achieve the full potential of human ES cells as a tool for both therapeutic development and basic research. It will enable the repairing of faulty genes in patient specific ES cells or induced pluripotent stem (iPS) cells, thereby speeding up the development of ES cell based therapeutic strategies. Further, the ability to inactivate or modify gene function in human ES cells will permit scientists to define roles of genes in self-renewal or differentiation of these cells, which is crucial for a clear understanding of the ES cell biology and ultimately developing better diagnostics and therapeutic tools.
In aim 1, this proposal seeks to improve the efficiency of homologous recombination in human embryonic stem cells (hESCs) by comparing a conventional electroporation approach with the use of adeno-associated virus (AAV)-mediated DNA delivery. The investigators will also examine the effects of homology arm length and of isogenic DNA on gene targeting frequency. In aim 2, the authors propose to use the optimum technique to target eight genes in hESC. They will add a tag that can be used for immunoprecipitation to four transcription factors, and they will place a fluorescent marker into the genomic location of four genes that are expressed during corticospinal or serotonergic development. Reviewers were moderately enthusiastic about this grant. The goals of the grant were considered important, the project was well-designed, and the principal investigator (PI) and main collaborator were described as strong. However, reviewers’ enthusiasm was curbed as the work seemed overly ambitious for a two-year grant, and there were concerns about the lack of commitment of the main collaborator. Reviewers expressed different opinions about the potential impact of the first aim of this proposal. All reviewers agreed that the AAV method was potentially exciting. However, some reviewers commented that the validation and comparison of the two methods for homologous recombination would be very important, whereas others felt that it was irrelevant. Furthermore, although one reviewer felt that the choice of targeted locus during the optimization studies was clever, others pointed out that a similar approach is being pursued by others. With regard to aim 2, reviewers commented that the cell lines generated would be valuable tools. The tagged transcription factors would greatly facilitate chromatin immunoprecipitation experiments and genome wide analysis of genetic regulatory networks - work that would clearly fall directly into the core interests of the PI’s laboratory. The reporter gene work would also generate a series of very useful reagents that could be utilized by the wider research community for studying hESC differentiation to neuronal cell types. However, the reviewers questioned the rationale for including the two disparate goals of aims 1 and 2 in the same application, especially given the amount of work involved in each one. Individual experiments were judged to be feasible, but the overall plan was considered too ambitious for a two-year grant. The first aim, to optimize homologous recombination in hESCs, received several minor criticisms. The rationale for committing resources to further optimize the gene targeting frequency using conventional methods (Aim 1a) was questioned, as one reviewer commented that the work had already been published by others. Investigating the use of AAV was considered more interesting. In terms of the preliminary data, reviewers were concerned that the description of the HUES9 clones containing targeting events at the specific locus the PI is studying did not involve karyotyping. As maintenance of a normal karyotype is likely to be considered an important feature of genetically modified hESCs, it would have been prudent to initiate these studies with lines that afforded this possibility. The preliminary data provided to support the second aim is sufficient to suggest that the applicants would eventually be able to generate the reagents that have been listed. However, the overall volume of proposed gene targeting experiments is large and there is good reason to think that only a fraction of the reagents would be generated within the time frame of the grant. Reviewers commented that the application might have fared better if only one of these projects had been proposed. The PI has strong experience in technology and genome mapping, has an impressive publication track record in the areas of whole genome analysis of genetic regulatory networks. This includes senior authorship on a number of papers in high impact journals. The main collaborator is a well-trained and very promising young neuroscientist, and brings needed expertise with ESC genetic modifications to the project. A final collaborator provides AAV expertise. Reviewers expressed concern that neither collaborator committed any percent effort to this project. Overall, reviewers considered this proposal to be of high impact, but reviewers doubted whether the work described could be accomplished within two years.