Characterizations of Cell Lineage Markers in the Bone Marrow Cells of Recloned GFP Pigs for Possible Use of Stem Cell Population

서지정보

ㆍ발행기관 : 한국동물번식학회 ㆍ수록지정보 : Reproductive & developmental biology / 35권 / 1호
ㆍ저자명 : Kwang-Wook Park, Sung Sik Choi, Dong Ho Lee, Hwang Lee, Seung Kyu Choi, Chang Sik Park, Sang Ho Lee

목차

ABSTRACT INTRODUCTION MATERIALS AND METHODS Animals Recovery of Bone Marrow Cells Cell Culture Determination of GFP Expression in BMCs Immunocytochemistry for Cell Lineages Reverse Transcription-Polymerase Chain Reaction (RTPCR)and PCR of Genomic DNA Western Blotting Analysis for GFP RESULTS Different Extent of GFP Expression among the ReclonedGFP Pigs Cellular Distribution of GFP Expressed in GFP-positiveCells Expressions of Cell Surface Markers, SSEA-1, SSEA-3and CD34 in GFP-positive Cells Expressions of Neural Cell Lineage Markers in GFPpositiveCells Verification of Insertion and Expression of GFP Genein the Tissues of Recloned Pigs DISCUSSION REFERENCES

영어 초록

Two piglets and one juvenile pig were used to investigate closely what types of cells express green fluorescent protein (GFP) and if any, whether the GFP-tagged cells could be used for stem cell transplantation research as a middle-sized animal model in bone marrow cells of recloned GFP pigs. Bone marrow cells were recovered from the tibia, and further analyzed with various cell lineage markers to determine which cell lineage is concurrently expressing visible GFP in each individual animal. In the three animals, visible GFP were observed only in proportions of the plated cells immediately after collection, showing 41, 2 and 91% of bone marrow cells in clones #1, 2 and 3, respectively. The intensity of the visible GFP expression was variable even in an individual clone depending on cell sizes and types. The overall intensities of GFP expression were also different among the individual clones from very weak, weak to strong. Upon culture for 14 days in vitro (14DIV), some cell types showed intensive GFP expression throughout the cells; in particular, in cytoskeletons and the nucleus, on the other hand. Others are shown to be diffused GFP expression patterns only in the cytoplasm. Finally, characterization of stem cell lineage markers was carried out only in the clone #3 who showed intensive GFP expression. SSEA-1, SSEA-3, CD34, nestin and GFAP were expressed in proportions of the GFP expressing cells, but not all of them, suggesting that GFP expression occur in various cell lineages. These results indicate that targeted insertion of GFP gene should be pursued as in mouse approach to be useful for stem cell research. Furthermore, cell- or tissue-specific promoter should also be used if GFP pig is going to be meaningful for a model for stem cell transplantation.

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