Modeling Disease in Human ESCs Using an Efficient BAC-Based Homologous Recombination System

Abstract

Although mouse models have been valuable for studying human disease, the cellular and physiological differences between mouse and human have made it increasingly important to develop more relevant human disease models for mechanistic studies and drug discovery. Human embryonic stem cells (hESCs), which can undergo unlimited self-renewal and retain the potential to differentiate into all cell types, present a possible solution. To improve the efficiency of genetic manipulation of hESCs, we have developed bacterial artificial chromosome (BAC) based approach that enables high efficiency homologous recombination. By sequentially disrupting both alleles of ATM or p53 with BAC targeting vectors, we have established ATM(-/-) and p53(-/-) hESCs as models for two major human genetic instability syndromes and used the generated cells to reveal the importance of p53 in maintaining genome stability of hESCs. Our findings suggest that it will be feasible to develop genetically modified hESCs as relevant human disease models.

Analysis of microRNAs in a knock-in hESC line expressing epitope-tagged AGO2

“…The homologous recombination events were confirmed by ligationmediated inverse PCR as described previously (Figure 1 (b)) (Song et al 2010). We obtained 2 positive clones out of 18 G418-resistant clones.…”

“…To modify the AGO2 locus to express a FLAG-tagged version of AGO2 (FLAG-AGO2), a BAC-mediated targeting strategy was employed as previously described (Song et al 2010). The targeting construct was made by exploiting homologous recombination machinery incorporated into bacterial cells (Copeland et al 2001).…”

Novel approaches to in vitro transgenesis

“…One of the key components of the development is the generation of the homologous recombination cassette, usually several kilobases in length. Recently, Song et al (2010) exploited the coverage of the human genome by BACs to generate a recombination cassette with extended homology arms for the creation of p53 knockout human ES cells. This BAC-based targeting approach had several advantages, such as the introduction of large DNA sequences, leading to a high efficiency of homologous recombination in various genetic backgrounds.…”

“…This BAC-based targeting approach had several advantages, such as the introduction of large DNA sequences, leading to a high efficiency of homologous recombination in various genetic backgrounds. This technique avoided the more complex and technically difficult cloning strategies by using BAC recombineering technology to generate the recombination cassette (Song et al 2010). One reported drawback of this BAC-based targeting approach was the difficulty in confirming that the homologous recombination event had occurred.…”

Find and replace: editing human genome in pluripotent stem cells

“…A few other research groups also successfully obtained gene-modified cell lines with this conventional approach, nevertheless with extremely low efficiency (Urbach et al, 2004;Costa et al, 2007;Irion et al, 2007;Davis et al, 2008;Di Domenico et al, 2008;Bu et al, 2009;Kamei et al, 2009;Ruby and Zheng, 2009;Xue et al, 2009;Buecker et al, 2010;Fischer et al, 2010;Goulburn et al, 2011). To overcome technical limitations, novel gene editing strategies have been developed to enhance targeting efficiency in hPSCs, such as bacterial artificial chromosome (BAC) (Song et al, 2010), adenoassociated virus vector (AAV) (Khan et al, 2010;Asuri et al, 2011), zinc finger nuclease (ZFN) (Lombardo et al, 2007;Hockemeyer et al, 2009;Zou et al, 2009), transcription activator-like effecter nuclease (TALEN) (Cermak et al, 2011;Hockemeyer et al, 2011;Miller et al, 2011;Mussolino et al, 2011;Zhang et al, 2011), and helper-dependent adenoviral vector (HDAdV) (Suzuki et al, 2008;Li et al, 2011;Liu et al, 2011b). Here we focus on comparing cons and pros of the above novel techniques to explore their potential for efficient and safe gene targeting in hPSCs.…”