研究主題.IV.建立與研究基因轉殖與剔除小鼠(English version)

My long term research goal is to study the pathogenesis of complex human diseases with an emphasis on solving the “cause or consequence” puzzle using mouse genetics as a tool. Studies in human genetics have been fruitful in identifying potential gene(s) underlying the development of human diseases. However, a potential problem of human studies dealing with complex diseases arises when a mutation is found in only a limited number of individuals or families. The concurrent inheritance of other factors common to affected individuals and their relatives can influence the phenotype and make it difficult to establish a truly causative link between the mutation and the observed phenotype. Mouse models under a defined genetic background, a uniform diet, and a controlled environment offer a unique avenue to study the causative role of a gene and its involvement in complex human diseases.

As a first step for this process, I needed to be trained in utilizing mouse genetics as a tool to dissect complex diseases. I chose Dr. Nobuyo Maeda and Dr. Oliver Smithies’ Laboratory, which has pioneered the use of gene targeting in developing animal models for human diseases. In the past six years, I generated three independent mouse lines designed to study the relationship between a transcription factor, PPARg, and a common complex disease, metabolic syndrome. The significance of my work was recognized by American Heart Association by awarding me a Predoctoral Fellowship to pursue these studies.

In the first part of my work, I generated a mouse model with a clinically relevant PPARg point mutation. This mutation was identified in patients with severe insulin resistance, hypertension, and lipodystrophy. To establish a causative link between the PPARg mutation and the observed phenotypes in humans, I introduced the same mutation into the comparable mouse gene and assessed effects of the mutation on the metabolic state of the mice. Like humans, mutant mice have elevated blood pressure. They also display abnormal fat distribution, although the pattern of fat distribution in mice differs from that in humans. However, unlike humans, they maintain normal insulin sensitivity. Thus this mutation alone is sufficient to cause abnormal fat distribution and hypertension but not insulin resistance in mice. I published these results in JCI 114:240-249, 2004. This model has led to the important finding that PPARg is a critical factor in determining where body fat is stored. The importance of this finding is underscored by my being invited to write a review in Trends in Cardiovascular Medicine (TCM 15:81-85, 2005).

The second part of my work had two aims. First, to develop a unique system to test a critical assumption of human polymorphic associations in disease; namely that subtle changes in a particular gene are the major contributing factors of quantitative trait analysis. To do this, I was instrumental in developing a novel technique that can alter the expression in mice of chosen genes over a 10-fold range while retaining their chromosomal location and transcriptional controls. I am a co-author of this paper (Developmental Cell 6:597-606, 2004) describing this novel method. For the subsequent aim, I applied this general hypothesis specifically to PPARg-related changes in metabolic syndrome. I generated multiple mouse models with a spectrum of PPARg levels to test whether subtle change in PPARg levels is the basis leading to metabolic syndrome. The future work regarding this part is proposed under my research directions.

In addition to these “well-planed” projects, I also characterize an unexpected finding during my PhD/postdoc research. I identified a de novo mutation in the Pmca2 gene that arose in ES cells during culture. The reason we can spot this mutation is because it is tightly linked to the Pparg genetic modification we introduced and it causes severe neurologic phenotypes, ataxia. Although this cannot account for an exiting finding, I still comprehensively completed this small story and published in Mammalian Genome 17, 716-722, 2006.

For the next stage of my career, I plan to initiate research work that will focus on dissecting the contribution of inflammation to metabolic syndrome. Metabolic syndrome is usually associated with the increases in a series of pro-inflammatory cytokines, implicating the connection between inflammation and metabolic syndrome. Two recent studies (JCI 112:1796-1808, 2003 and JCI 112:1821-1830, 2003) showed that macrophages infiltrate adipose tissues in the states of obesity. Several critical questions are raised here. What are the implications of macrophage infiltration into adipose tissue in obesity? Is this a cause or consequence of obesity? Does this inflamed adipose tissue lead to development of insulin resistance? As a framework against which to investigate these questions, I propose a testable hypothesis. I hypothesize that systemic or local (such as muscle, adipose tissue, liver, and pancreas) increase of inflammatory responses causes metabolic syndrome.

To test this hypothesis, I plan to generate a knock-in mouse model, in which inflammatory responses can selectively be increased in the chosen tissue (or cell). I will use this mouse model to study the effect of “inflamed” tissue in the development of metabolic syndrome.