BRL started on June 1, 2010, as a core of the research project gDevelopment of bioresources for the next generationh (2007.4-2012.3) funded by The Ministry of Education, Science and Culture of Japan, Grants-in-Aid for Scientific Research. Its mission is to create a novel library of disease model cells focused on diabetes. Gene modification is performed mainly by RNAi method to produce cell lines with different knockdown (KD) rates such as 80%, 60%, or 25% inhibition. The production of knockout (KO) cell lines is also challenged as well. These cell lines should be useful for the quantitative analysis of multi-factor induced diseases such as diabetes, cancer, and other adult diseases. A multi-factor induced disease is thought to be not caused by KO of a specific one gene but by simultaneous KD of multiple genes at different KD rates.
1. Gene modified ES cell lines relevant to diabetes
The gene modification performed so far with mouse ES cells by our laboratory is the KD of single or double genes selected from 8 genes (Pdx-1, IRS-1, Kir6.2, IRS-2, GK, SHP, Hnf-1?, Hnf-1?) by RNAi method, the KO of GK by gene targeting method, and over expression (OE) of single gene. Thus produced ES cell lines make a diabetes model cell library. These cell lines are differentiated into insulin generating cells or used to produce gene modified mice.
2. Mice in which diabetes related genes are modified
GK knockout mouse was produced using GK knockout ES cell. Knockout means the deletion of a single strand (hetero KO) or double strand (homo KO) of GK gene from the genome. In hetero KO, the GK gene expression level is expected to be 50% of the control.
3. Element technologies to evaluate bioresource function
Real time analysis of the gene expression in target single-cell is performed by femtoinjection of specific regulators and probes. Regulators include OE vectors, shRNA expression vectors, gene product proteins, and antibodies. Probes include a probe vector constructed with a target gene promoter and a fluorescent reporter gene such as GFP.
