Phenotype analysis of Ndst4 knockout mice and preparation of mouse Ndst4 antisera
Date Issued
2012
Date
2012
Author(s)
Lo, Wei-Chen
Abstract
The development of colorectal cancer originates from accumulation of many genetic defects, including activation of oncogenes and deletion of tumor suppressor genes. Our previous study found a putative tumor suppressor gene, NDST4, at chromosome 4q26 in colorectal cancer. So, we produced a Ndst4 knock out (Ndst4 KO) mouse to study the probable physiological functions of NDST4. In this thesis, there are two parts of investigation. First, we want to examine whether there are phenotype differences between Ndst4 KO and wild type (WT) mice. In this part, we used Southern blotting and multiplex PCR to identify the genotype of Ndst4 KO mice. Then, we used RT-PCR to check the tissue spectrum of Ndst4 RNA expression. We used the organ (Brain) with higher RNA expression to confirm that Ndst4 KO mice have no Ndst4 RNA expression. Finally, we conducted the modified-SHIRPA, complete blood count (CBC), biochemistry and histology analyses to compare Ndst4 KO and WT mice. In the modified-SHIRPA, CBC and biochemistry, we did not get significantly different results. However, in the histology analysis, we found that there were more goblet cells in proximal colon, and small intestine had morphological change in Ndst4 KO mice. Second, we want to produce mouse Ndst4 antisera. Because Ndst family has four members that have high similarity in protein sequences. So, we aligned all of the Ndst protein sequences by CLC sequence view 6 software, and found out a region with lower similarity. Then, we selected the sequences selected to perform B-epitope prediction. According to the information obtained, we decide to use 40 to 226 amino acids as immunogen. We used E coli system to express the target protein, and then performed protein purification. Finally, we used Balb/C mice to perform antigen immunization, and used Western blotting to determine titers and specificity of mouse antisera. All of the mouse antisera could recognize mouse Ndst4 antigen. In addition, 2 mouse antisera also could recognize human NDST4 (hNDST4) antigen. At present, we have got mouse antisera against mNdst4 and hNDST4 that could be applied at NDST4-associated study in the future.
Subjects
Colorectal cancer
Knock out mice
Phenotype
monoclonal antibody
SDGs
Type
thesis
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