Developing dopaminergic reporter hESC lines by homologous recombination

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01028
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
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.
Progress Report: 
  • Stem cells have enormous potentials to both regenerate themselves and differentiate into different mature cell types, such as lung cells or liver cells. The ability to manipulate stem cell genomes is useful for both understanding stem cells at a fundamental level as well as for practical and therapeutic purposes, such as regenerative medicine. The focus of this research project is to develop a genetic tool for gene editing of human embryonic stem (hES) cells with high precision and efficiency. An engineered delivery system has been constructed and tested for their ability to deliver a genetic scissor and/or a donor DNA. We found that this system can accomplish targeted disrupt of desired genes in the genome with high efficiency (30%) and great accuracy. Our experiments also confirmed that such a system can efficiently mediate specific gene addition to a predetermined target site of hES cells. The modified hES cells maintained their self-renewal and pluripotent state of the stem cells. We have also evaluated an mRNA display technique for designing new genetic scissors specific for genome sites of stem cell interests. We showed that such a technique could be adapted to generate scaffold scissors capable of binding to target DNAs. Our experiments further demonstrated the feasibility of utilizing this powerful method to establish a library of ten trillion scissor proteins for selections to identify stem cell-specific binders.
  • Human embryonic stem (hES) cells are renewable cell sources that have potential applications in regenerative medicine. The development of technologies to produce permanent and site-specific genome modifications is in demand to achieve future medical implementation of hES cells. We show that a baculoviral vector (BV) system carrying zinc finger nucleases (ZFNs) can successfully modify the hES cell genome. BV-mediated transient expression of ZFNs specifically disrupted the CCR5 locus in transduced cells and the modified cells exhibited resistance to HIV-1 transduction. To convert the BV to a gene targeting vector, a DNA donor template and ZFNs were incorporated into the vector. These hybrid vectors yielded permanent site-specific gene addition in both immortalized human cell lines (10%) and hES cells (5%). Modified hES cells were both karyotypically normal and were pluripotent. These results suggest that this baculoviral delivery system can be engineered for site-specific genetic manipulation in hES cells. We have also validated the mRNA display technology for designing new ZFNs for targeting transcriptional factors involved in controlling hES cell self-renewal and differentiation.
  • In our previous progress report, we detailed our efforts targeting the human beta-globin gene with zinc finger nucleuses (ZFNs). Our goal was to correct a mutation that results in sickle cell anemia, the most common inherited blood disorder in the United States, affecting ~80,000 Americans. Two recent published reports evaluating the specificity of ZFNs demonstrates the importance of designing highly specific ZFNs to avoid off-target effects. Thus, we have reevaluated our choice of target, our library design, and our mRNA display strategy for designing beta-globin-targeted ZFNs. In addition, we carefully evaluated our protocol for differentiating human embryonic stem cells (hESCs). By combining with growth factors, the undifferentiated hESCs can be directly differentiated into three germ layers through EB formation. The in vivo results also confirmed that hESCs gown in a scaffold could support the growth and differentiation of undifferentiated hESCs into different germ layers by providing physical environment for the interaction of hESCs with host tissues.

© 2013 California Institute for Regenerative Medicine