In the past, naturally-mutated animals were used to elucidate the mechanisms of various diseases. In the "post-genome" project era, however, genetically manipulated animals that can serve as animal models for human diseases play a key role in such investigations. Our laboratory assists other research facilities in generating such genetically manipulated animals, as shown by our web page (http://kumikae01.gen-info.osaka-u.ac.jp/EGR/index.cfm). This objective is undertaken in collaboration with the Animal Resource Center for Infectious Diseases.
We were the first in the world to produce a genetically altered "green mouse" that glows in the dark. These mice are highly useful for many types of research, including stem cell transplantation and regeneration. By utilizing these animals, we showed that the sex of murine embryos can be determined at the preimplantation stage. These mice have been used to study the fertilization process (Fig. 1) (3) and the sex determination mechanism in germ cells.
We are also interested in the fertilization process in terms of self-nonself recognition. By utilizing homologous recombination technology, we showed protein IZUMO1 as the first sperm factor that plays an essential role in the fusion of sperm with eggs; we also found recently that the sperm protein SPESP1 is needed for the production of fully fusion-competent sperm (Figs. 2 and 3) (1, 2).
Fig. 1. A transgenic mouse line whose sperm express green fluorescent protein (GFP) in their acrosome and red fluorescent protein (RFP) in their mitochondria. This development makes it possible to obtain live images of sperm in vivo (3). |
Fig. 2. Izumo1 KO sperm accumulate in the perivitelline space of the egg because they cannot fuse with the egg (1). |
Fig. 3. The membrane of the entire equatorial segment area is detached in almost all acrosome-reacted Spesp1-deficient sperm (right) (2).
In addition to our studies on the sperm-egg interaction, we are studying the roles the placenta plays in feto-maternal immune tolerance. Since we believe gene functions are best observed in live animals, we sought a method that would permit genetic manipulation of the placenta. We were eventually successful in developing a Lentiviral vector-based method that mediates the genetic manipulation of the placenta without affecting the embryos (Fig. 4) (4).
We are also interested in understanding the biological function of non-coding RNA such as miRNA. The miRNA knock-out technique is being used to identify the roles miRNA plays in live animals.
Fig. 4. Placenta-specific gene manipulation. GFP-transgene expression at E14.5 after gene manipulation. Shown are embryos in mice with (left) an untransduced placenta, (middle) a placenta that had been subjected to the normal transgenic procedure, and (right) a placenta that had been altered by our newly developed method for placenta-specific genetic manipulation (4).
Miwa’s group is using genetically manipulated animals to investigate the molecular biological mechanisms that are involved in human diseases, especially cardiovascular diseases. To understand the cellular and molecular aspects of vascular smooth muscle (SM) cell growth in atherosclerotic plaques, we characterized the transcriptional mechanisms of one SM-specific gene, the SM alpha-actin (SmaA) gene. Since SmaA is also expressed in many tissues during acute inflammation, we are currently analyzing its gene expression system and its functional roles (Fig. 5) (5). We are also analyzing the molecular pathogenic mechanisms of diastolic heart failure by using a Dahl salt-sensitive rat model. Specifically, we are currently investigating how the endothelin and renin-angiotensin systems participate in the excessive hypertrophy and fibrosis that eventually leads to diastolic heart failure.
Fig. 5. The human SM alpha-actin promoter (left) expresses in the embryonic aorta. However, point mutations in the enhancer region of this promoter, including the -1M (center) and 4M (right) mutations, eliminate this specific expression pattern (5).