| Budget Amount *help |
¥7,900,000 (Direct Cost: ¥7,900,000)
Fiscal Year 1994: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1993: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Research Abstract |
while a numbers of key growth factors that regulates hematopoiesis have been identified and some of them are currently used for clinical purpose, how bone marrow microenvironment that presents those signals to the hematopoietic progenitors is constructed remains totally obscure. The purpose of the present study is to produce model mice in which the hematopoiesis supporting cells are easily distinguished from other stromal cell component. For this purpose, we attempted to produce transgenic mice that bear LacZ marker gene under the control of the transcription regulating region of KL gene that is required for the self-renewal of all hematopoietic progenitors. We have cloned genomic KL gene and 10kb upstream from the transcription initiation site was analyzed by CAT assay using hematopoiesis supporting stromal cell line ST2. We found that 35bp upstream of TATA-box is sufficient for assigning CAT-gene expression in this cell line. However, this region has no activity when it was used for
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making transgenic mice. This indicates that other cis-regulatory regions might be essential for the regulation of KL-gene in vivo. Hence, we then introduced LacZ gene hocked to 2kb , 6kb and 10kb upstream region of KL gene into fertilized eggs and made transgenic mice . As expected, some transgenic mice bearing 2kb or 6kb constructs showed tissue specific expression of LacZ gene in hair follicles, gut pacemaker cells and neurons where KL is expressed. However, with regard to 42 strains thus far produced , none showed expression in the hematopoietic tissues, except one strain in which LacZ expression was observed in osteoblast cells. These results indicates that KL-expression in hematopoietic microenvironment is mediated by yet unknown cis-regulatory elements of KL-gene. Nevertheless, our original purpose to make model mice by this strategy failed commpletely.We think that employment of newly-emerged technology like gene-knock-in method would be more helpful to accomplish our original purpose.
In addition to the study in the above direction, we have attempted to make monoclonal antibodies against growth factor receptors that is essential for hematopoiesis.In addition to hitherto established anti-c-kit mAb, we have succeeded to produce mAbs against IL-7R,c-fms and flk2/flt3. Though our studies using these mAbs have not completed, our anti-IL-7R mAb contributed to demonstrate that IL-7 Plays an essential role in the pathophysiology of X-chromosome linked combined severe immunodeficiency. We expect that other mAb will be also useful to dessect the tissue structure of hematopoietic organs. Less
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