Institute of Genetics

Dr Chris Denning

Precise genetic manipulation of the mammalian genome by gene targeting and through emerging technologies such as RNA interference (RNAi) are crucial to furthering our understanding of biological systems and to developing models of human disease1.

Cloning technology for disease resistant animals

While it has been possible for many years to manipulate the genome of mice by gene targeting via the embryonic stem (ES) cell route, lack of suitable germline competent ES cells in any other mammalian species has prohibited wider application of the technology. Cloning by somatic cell nuclear transfer (SCNT; Fig. 1) provides an alternative route to cell-based transgenesis2. We have previously used this technology to produce PrP+/- lambs (Fig. 2), which have one allele of the gene encoding prion protein inactivated by gene targeting3. Eliminating this gene, which is involved in spongiform encephalopathies such as scrapie in sheep and BSE in cattle, will be important in the biotechnology sector where with an increasing use of livestock as ‘biorectors’ for human recombinant proteins it would be preferable to use animals that have a guaranteed prion-free genetic background. In collaboration with Merial Ltd (USA) and Viagen Inc (USA), we are now generating PrP knockout cattle.

Fig. 1 The process of somatic cell nuclear transfer (SCNT) involves removal of metaphase II chromosomes from oocytes derived from superovulated animals. A donor somatic cell, which can be genetically modified in culture, is placed under the zona pelucida of the enucleated oocyte. Fusion of the membranes of donor cell and recipient oocyte, and activation of embryo development is commonly initiated by electrical pulses.

Fig. 2 By transferring gene targeted embryos produced by SCNT to surrogate ewes, we have previously reported birth of PrP+/- lambs3.

Human embryonic stem cells

The ability of mouse ES cells to contribute to all tissues within the developing embryo prompted efforts to isolate equivalent cells from the inner cell mass of human embryos. First achieved in 1998 by Thomson and colleagues4, hES cells offer many potential opportunities: new approaches to the study of human embryonic development; creation of in vitro human disease models for basic research, drug discovery and toxicology; cell therapy and so on.

We are using the unique properties of hES cells to provide a novel in vitro model of Duchenne muscular dystrophy (DMD). This X-linked lethal genetic condition affects ~1 in 3000 new born males, confining them to a wheel chair by puberty and ultimately leading to death from respiratory or cardiac failure in their early twenties5. Cardiac involvement is often seen in patients, presenting as electrocardiogram abnormalities, arrhythmia and cardiomyopathy, usually resulting in heart failure6. Furthermore, in a study, which included children as young as four, ultrasonic tissue characterisation analysis showed virtually all patients were at risk of developing dilated cardiomyopathy7. Although classified as a muscular dystrophy, DMD patients also often present with mental retardation, and gastric, retinal and kidney disorders8, consistent with the wide range of tissue types in which dystrophin is expressed.

While it is known DMD is caused by mutations in the dystrophin gene that eliminate protein production and / or function, the molecular consequences are relatively poorly understood. This is exacerbated by limited availability of many of the patients’ tissues, including cardiac and neural tissue. Furthermore, disruptions of two genes in mice are required before the disease starts to more accurately model DMD in humans9. Dystrophin forms part of the large dystrophin associated protein complex (DAPC), where it appears to engage structural and signaling roles8. However, the exact nature of particularly the signaling roles is far from clear and requires further investigation. Thus, we will develop a novel in vitro approach to studying DMD by using gene targeting in hES cells to recapitulate a naturally occurring dystrophin mutation coupled with directed differentiation to affected lineages, including cardiomyocytes. Assessment of DMD and non-DMD differentiated lineages will be carried in collaboration with Professor Kay Davies (University of Oxford).

We are culturing the commercially available hES line, BGN.01, in collaboration with Dr. Lorraine Young (School of Human Development), by manual and bulk passaging methods on mouse embryo fibroblast (MEF) feeders or in feeder-free conditions on matrigel supplemented with conditioned medium from MEFs. Under these conditions, the hES cells retain the expected surface marker characteristics of the undifferentiated state (Fig. 3). Furthermore, hES cells grown in feeder-free conditions can be readily transfected with short-interfering (si)RNA (Fig. 4), a route we are using to improve directed differentiation, and plasmid DNA, which will be essential for achieving gene targeting.

Fig. 3 BGN.01 hES cells cultured by manual or bulk culture methods such as dispase, collagenase, cell dissociation buffer or trypsin on MEF feeders or in feeder-free conditions (shown in figure) on matrigel supplemented with MEF conditioned medium react appropriately with markers associated with the undifferentiated state. By contrast to hES cells, mouse ES cells are SSEA-1+ve, SSEA-4-ve, TRA-160-ve and TRA-181-ve.

Fig. 4 Genes involved in epigenetic regulation (histone deacetylase 1; hDAC1) or potentially the undifferentiated state (signal transducers and activators of transcription-3; STAT-3) in hES cells can be knocked down singly or in combination by short interfering (si)RNA technology.

The BGN.01 hES cell line can be placed into non-adherent suspension culture to induce differentiation via embryoid bodies (EBs) to representatives of all three germ layers, including beating cardiomyocytes (Fig. 5). Cardiac differentiation of hES cells is stimulated by co-culture with the induce cell line END-2, a P19 EC subline10 and we are collaborating with Professor Christine Mummery (Hubrecht Lab, Utrecht, the Netherlands) to further investigate this approach using BGN.01 hES cells.

Human ES cell-derived cardiomyocytes respond to drugs known to stimulate or suppress beat rate and express cardiac markers (Fig 5). Further, they can be dissociated and subjected to single-cell elecrophysiological analysis by patch clamping. Through collaboration with Professor Ian Hall (Division of Medicine, Therapeutics and Molecular Medicine) and co-supervision of a PhD student (Gareth Goh), we are characterizing the dynamics of ion channel presentation during differentiation of BGN.01 towards the cardiac lineage.

Fig. 5 Formation of embryoid bodies by suspension culture of BGN.01 hES cells stimulates spontaneous differentiation to multiple lineages, including beating cardiomyocytes (A). Typical of cardiac cells, the beating areas are stimulated by ?-adrenergic receptor blockers (e.g. isoprenaline) and inhibited by agonists of muscarinic acetylcholine receptors (e.g. carbachol) (B). Cardiac specific gene expression can also be detected by RT-PCR (C).

References:

1) Denning C & Priddle H (2003) New frontiers in gene targeting and cloning: success, application and challenges in domestic animals and human embryonic stem cells. Reproduction 126, 1-11.

2) Campbell KHS et al., (1996). Sheep cloned by nuclear transfer from a cultured cell line. Nature 380, 64-66.

3) Denning C et al., (2001). Deletion of the ?(1,3)galactosyl transferase (GGTA1) and prion protein (PrP) genes in sheep. Nature Biotech. 19, 559-62.

4) Thomson JA et al., (1998). Embryonic stem cell lines derived from human blastocysts. Science 282 1145-7.

5) Emery AEH (1993). Duchenne Muscular Dystrophy, 2nd Ed. (Oxford: Oxford University Press).

6) Nakamura A et al., (2001). Activation of calcineurin and stress activated protein kinase/p38-mitogen activated protein kinase in hearts of utrophin-dystrophin knout mice. Neuromuscular Disorders. 11, 251-9.

7) Giglio V et al., (2003). Ultrasound tissue characterization detects preclinical myocardial structural changes in children affected by Duchenne muscular dystrophy. J. American College of Cardiol. 42, 309-16.

8) Rando A (2001). The dystrophin-glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies. Muscle & Nerve. 24, 1575-1594.

9) Deconinck AE et al., (1997). Utrophin-Dystrophin-Deficient Mice as a Model for Duchenne Muscular Dystrophy. Cell 90, 717–727.

10) Mummery C et al., (2003). Differentiation of Human Embryonic Stem Cells to Cardiomyocytes: Role of Coculture With Visceral Endoderm-Like Cells. Circulation. 107, 2733-2740.