Generation of Transgenic Mice and Pigs Harboring the DsRed-Monomer Reporter Gene and Characterization of Potentials for Biomedical Applications
Date Issued
2014
Date
2014
Author(s)
Chou, Chih-Jen
Abstract
Fluorescent proteins provide much more powerful targets to track gene expression and cellular behaviors in the study of model organisms. These proteins have served as invaluable tools fitting to those purposes related to genetic engineering studies for many of the coming years. The monomeric red fluorescent protein (RFP) is a long wavelength of fluorescent proteins，it expresses specific molecular characteristics, such as: rapid maturation at 37°C, brighter and lower energy excitation spectrum, and less automonous fluorescence of animal tissues etc. Base on the fact that this molecule behavior to be rapid maturation, the RFP has since been used particulars for tracking target in the recent years. The purpose of this study has been focused to is generate transgenic mice and pigs harboring the DsRed-Monomer reporter gene and to evaluate the potentials for biomedical applications. To meet purposes described above, the initial studied were made to generate the DsRed-monomer transgenic mouse and pig by means of pronuclear microinjection of the transgene driven by the cytomegalovirus CMV enhancer/chicken beta-actin promoter. Transgenic mice and pigs harboring a gene of monomeric red fluorescent protein were generated and further confirmed to be transmitted their modified genome into their offspring. Both of the founder mice and/or pigs were further comfirmed to be able to transmit their modified genotype into their first generation, with their germ-line transmission rates arroud 52.72% (58/110) and 43.59% (17/39) in these transgenic lined mice and pigs respectively. These transgenic mice and/or pigs appeared to be ubiquitously expressed their red fluorescent protein found in those sampling organs and/or tissues (including： brain、 eye、 tongue、 heart、 lung、 liver、 spleen、 pancreas、 stomach、 small intestine、 large intestine、 kidney、 testis、 uterus、 ovary、 muscle and hoofs etc.) were all microscopically confirmed by excitation light wavelength and filter (excitation 520-540 nm, emission 580-650 nm). Furthermore, such expression was reconfirmed by histological assay and western blot analyses of each various tissues. In the respect of histopathology assay, the mouse tissue sections dissected from organs or tissue (including： brain、 eye、 tongue、 heart、 lung、 liver、 spleen、 pancreas、 stomach、 small intestine、 large intestine、 kidney、 testis、 uterus、 ovary、 muscle and eye etc.) of wild-type and/or RFP transgenic mice, the results revealed the following distinct histological discrepancy in pancreases of transgenic mice with additional intra-cytoplasmic eosinophilic threadlike materials in acinar cells compared with those sample for the wild-type mice. Both of those values related to hematology, serology and/or plasma biochemistry value appeared to be important parameter(s) for further assess emendation of physical conditions and animal health. The transgenic mice were minor affected in these parameters relate to lymphocytes、 monocytes、 alanine aminotransferase (ALT) and aspartate aminotransferase (AST)， when comparisons were made these found in the wild-type mouse. However, there was no significant differences found between these of control pigs and Ds-red bearing transgenic pigs when histology、blood examinations and also the computed tomography were conducted. Furthermore, when progenitor stem cells derived from those of amniotic fluid of Ds-red bearing fetuses at day 70 of pregnant sows. These cells cells confirmed being able to be could differentiated into adipocyte、osteoblast even the chondrocyte in vitro. In addition, results generated affect the FACS (fluorescence-activated cell sorting) assessment indicate that these cells have been equipped with strong potential to express CD44, CD90 while not to express either CD4a and/or CD31 gene(s). Taken together, DsRed-Monomer transgenic mice and pigs were successfully produced, and confirmed the RFP expression on all of the sampling organs and transmitted the phenotype to their offspring. These red fluorescent mice and pigs can serve as a host for other fluorescent-labeled cells in order to study cell-microenvironment interactions, and can provide optimal red-fluorescent-labeled cells and tissues for research in developmental biology, regenerative medicine, and xenotransplantation.
Subjects
DsRed-monomer
red fluorescence protein
transgenic mice
transgenic pigs
Type
thesis