Journal of Genetics and Genomics

Beyond Mice: Genetically Modifying Larger Animals to Model Human Diseases

Xiao-Jiang Li, Wei Li

2012, 39(6): 237-238. doi: 10.1016/j.jgg.2012.05.006

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Abstract:

Influence of Species Differences on the Neuropathology of Transgenic Huntington's Disease Animal Models

Xiao-Jiang Li, Shihua Li

2012, 39(6): 239-245. doi: 10.1016/j.jgg.2012.05.002

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Abstract:
Transgenic animal models have revealed much about the pathogenesis of age-dependent neurodegenerative diseases and proved to be a useful tool for uncovering therapeutic targets. Huntington's disease is a well-characterized neurodegenerative disorder that is caused by expansion of a CAG repeat, which results in expansion of a polyglutamine tract in the N-terminal region of huntingtin (HTT). Similar CAG/glutamine expansions are also found to cause eight other neurodegenerative diseases that affect distinct brain regions in an age-dependent manner. Identification of this CAG/glutamine expansion has led to the generation of a variety of transgenic animal models. Of these different animal models, transgenic mice have been investigated extensively, and they show similar neuropathology and phenotypes as seen in their respective diseases. The common pathological hallmark of age-dependent neurodegeneration is the formation of aggregates or inclusions consisting of misfolded proteins in the affected brain regions; however, overt or striking neurodegeneration and apoptosis have not been reported in most transgenic mouse models for age-dependent diseases, including HD. By comparing the neuropathology of transgenic HD mouse, pig, and monkey models, we found that mutant HTT is more toxic to larger animals than mice, and larger animals also show neuropathology that has not been uncovered by transgenic mouse models. This review will discuss the importance of transgenic large animal models for analyzing the pathogenesis of neurodegenerative diseases and developing effective treatments.

Transgenic Nonhuman Primate Models for Human Diseases: Approaches and Contributing Factors

Yongchang Chen, Yuyu Niu, Weizhi Ji

2012, 39(6): 247-251. doi: 10.1016/j.jgg.2012.04.007

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Nonhuman primates (NHPs) provide powerful experimental models to study human development, cognitive functions and disturbances as well as complex behavior, because of their genetic and physiological similarities to humans. Therefore, NHPs are appropriate models for the study of human diseases, such as neurodegenerative diseases including Parkinson's, Alzheimer's and Huntington's diseases, which occur as a result of genetic mutations. However, such diseases afflicting humans do not occur naturally in NHPs. So transgenic NHPs need to be established to understand the etiology of disease pathology and pathogenesis. Compared to rodent genetic models, the generation of transgenic NHPs for human diseases is inefficient, and only a transgenic monkey model for Huntington's disease has been reported. This review focuses on potential approaches and contributing factors for generating transgenic NHPs to study human diseases.

Pluripotent Stem Cells Models for Huntington's Disease: Prospects and Challenges

Richard L. Carter, Anthony W.S. Chan

2012, 39(6): 253-259. doi: 10.1016/j.jgg.2012.04.006

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Pluripotent cellular models have shown great promise in the study of a number of neurological disorders. Several advantages of using a stem cell model include the potential for cells to derive disease relevant neuronal cell types, providing a system for researchers to monitor disease progression during neurogenesis, along with serving as a platform for drug discovery. A number of stem cell derived models have been employed to establish in vitro research models of Huntington's disease that can be used to investigate cellular pathology and screen for drug and cell-based therapies. Although some progress has been made, there are a number of challenges and limitations that must be overcome before the true potential of this research strategy is achieved. In this article we review current stem cell models that have been reported, as well as discuss the issues that impair these studies. We also highlight the prospective application of Huntington's disease stem cell models in the development of novel therapeutic strategies and advancement of personalized medicine.

Retake the Center Stage – New Development of Rat Genetics

Sushuang Zheng, Kindiya Geghman, Sushila Shenoy, Chenjian Li

2012, 39(6): 261-268. doi: 10.1016/j.jgg.2012.05.003

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The rat is a powerful model for the study of human physiology and diseases, and is preferred by physiologists, neuroscientists and toxicologists. However, the lack of robust genetic modification tools has severely limited the generation of rat genetic models over the last two decades. In the last few years, several gene-targeting strategies have been developed in rats using N-ethyl-N-nitrosourea (ENU), transposons, zinc-finger nucleases (ZFNs), bacterial artificial chromosome (BAC) mediated transgenesis, and recently established rat embryonic stem (ES) cells. The development and improvement of these approaches to genetic manipulation have created a bright future for the use of genetic rat models in investigations of gene function and human diseases. Here, we summarize the strategies used for rat genetic manipulation in current research. We also discuss BAC transgenesis as a potential tool in rat transgenic models.

Targeted Genome Editing by Recombinant Adeno-Associated Virus (rAAV) Vectors for Generating Genetically Modified Pigs

Yonglun Luo, Emil Kofod-Olsen, Rikke Christensen, Charlotte Brandt Sørensen, Lars Bolund

2012, 39(6): 269-274. doi: 10.1016/j.jgg.2012.05.004

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Recombinant adeno-associated virus (rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases. Several advantages, such as simple vector construction, high targeting frequency by homologous recombination, and applicability to many cell types, make rAAV an attractive approach for targeted genome editing. Combined with cloning by somatic cell nuclear transfer (SCNT), this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1, and breast cancer. This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination. We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts, which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.

Rapid and Cost-Effective Gene Targeting in Rat Embryonic Stem Cells by TALENs

Chang Tong, Guanyi Huang, Charles Ashton, Hongping Wu, Hexin Yan, Qi-Long Ying

2012, 39(6): 275-280. doi: 10.1016/j.jgg.2012.04.004

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Abstract:
The rat is the preferred animal model in many areas of biomedical research and drug development. Genetic manipulation in rats has lagged behind that in mice due to the lack of efficient gene targeting tools. Previously, we generated a knockout rat via conventional homologous recombination in rat embryonic stem (ES) cells. Here, we show that efficient gene targeting in rat ES cells can be achieved quickly through transcription activator-like effector nuclease (TALEN)-mediated DNA double-strand breaks. Using the Golden Gate cloning technique, we constructed a pair of TALEN targeting vectors for the gene of interest in 5 days. After gene transfection, the targeted rat ES cell colonies were isolated, screened, and confirmed by PCR without the need of drug selection. Our results suggest that TALEN-mediated gene targeting is a superior means of establishing genetically modified rat ES cell lines with high efficiency and short turnaround time.

Production of Brown/Yellow Patches in the SLC7A11 Transgenic Sheep via Testicular Injection of Transgene

Xin He, Hongtao Li, Zhiyong Zhou, Zongsheng Zhao, Wei Li

2012, 39(6): 281-285. doi: 10.1016/j.jgg.2012.04.005

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Abstract:
The gene, SLC7A11, which encodes the solute carrier family 7 member 11 (anionic amino acid transporter light chain, xCT), has been reported to be implicated in multiple processes such as in pheomelanin production, cell proliferation and migration, Kaposi's sarcoma herpesvirus (KSHV) entry into the host cells, learning and memory. Its involvement in cancer cell proliferation and metastasis has been widely studied. Its role in pheomelanogenesis is likely conserved in sheep. The full-length cDNA of sheep SLC7A11 was cloned from sheep skin fibroblasts for evaluating its role in regulating sheep coat color. The complete open reading frame of sheep xCT (sxCT) is 1512bp in length, encoding a 503 amino acid polypeptide. We explored its function on pheomelanogenesisin vitro and in vivo. In the melan-a non-agouti mouse melanocytes that mainly produce eumelanin, overexpressed sxCT reduced the content of eumelanin. Using a testicular injection transgenic method, sxCT-transgenic sheep were generated and exhibited patches of brown/yellow coat, suggesting that sxCT can be selectively expressed to increase the pheomelanin production in wool. Our studies suggest that testicular injection of transgene can be used to genetically modify sheep coat color.

Automated Behavioral Phenotyping Reveals Presymptomatic Alterations in a SCA3 Genetrap Mouse Model

Jeannette Hübener, Nicolas Casadei, Peter Teismann, Mathias W. Seeliger, Maria Björkqvist, Stephan von Hörsten, Olaf Riess, Huu Phuc Nguyen

2012, 39(6): 287-299. doi: 10.1016/j.jgg.2012.04.009

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Characterization of disease models of neurodegenerative disorders requires a systematic and comprehensive phenotyping in a highly standardized manner. Therefore, automated high-resolution behavior test systems such as the homecage based LabMaster system are of particular interest. We demonstrate the power of the automated LabMaster system by discovering previously unrecognized features of a recently characterized atxn3 mutant mouse model. This model provided neurological symptoms including gait ataxia, tremor, weight loss and premature death at the age of 12 months usually detectable just 2 weeks before the mice died. Moreover, using the LabMaster system we were able to detect hypoactivity in presymptomatic mutant mice in the dark as well as light phase. Additionally, we analyzed inflammation, immunological and hematological parameters, which indicated a reduced immune defense in phenotypic mice. Here we demonstrate that a detailed characterization even of organ systems that are usually not affected in SCA3 is important for further studies of pathogenesis and required for the preclinical therapeutic studies.

2012 Vol. 39, No. 6