HTm4 expression during B cell differentiation and the effects of HTm4 knockdown in myeloid cell differentiation

• Main Authors: Hsiu-Chuan Chien, 簡秀娟
• Other Authors: Jon Ko
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/69495644509362870977

碩士 === 國立陽明大學 === 生物藥學研究所 === 96 === The expression of HTm4 is confined to the tissues of hematopoietic origin and shown to be differentiation stage- and cell lineage-dependent. In the cascade of hematopoietic differentiation, HTm4 is detected consistently in B cell lineages with CD19 (+) phenotype, in some subpopulations but not all of myelocytic lineages with CD11b (+) phenotype, and none in cells with CD3 (+) marker, which is considered T cell lineage specific. Our previous data also showed that the ectopic expression of HTm4 in U937 cell brought about G0/G1 cell cycle arrest. Overall, HTm4 is affirmatively considered as a hematopoietic cell cycle regulator. Nonetheless, the involvement of HTm4 in the course of hematopoietic cell differentiation is currently unknown. To better define the expression patterns for HTm4 along the cascade of hematopoietic differentiation, we have constructed an EGFP knock in-HTm4 targeting vector for the procurement of an Embryonic stem cell (ESC) with one HTm4 allele containing an EGFP knock-in that is under the control of HTm4 promoter. So far we have isolated 350 ESC clones and determined all to be without the prospective EGFP knockin at the intended HTm4 genomic locus using genomic-PCR analysis. It is likely due to the low frequency of homologous recombination for HTm4 locus. Our future work will be to screen more ESC clones. Additionally, our preliminary data showed that upon induction of ESC targeted for B lineage differentiation, using OP 9 coculture system in the presence of Flt-3 ligand and IL-7, the expression of HTm4 mRNA is detected at day 12 after induction. This time-frame is coincided with the appearance of hematopoietic burst. The second part of this dissertation is to study the effect HTm4 on myeloid lineage differentiation. WEHI-3 cells with HTm4-knockdown, using siRNA technique, were procured, and the effects of HTm4-knockdown on the myelocytic differentiation of WEHI-3 were analyzed. When treated with all-trans retinoic acid and G-CSF, WEHI-3 was induced to undergo myelocytic differentiation. Two HTm4-knockdown WEHI-3 clones, siRNA-HTm4-61 #6 and #7 (81% and 92% efficacy, respectively) were used for the analysis. Both clones showed prominent increase in the promotion of myeloid differentiation using CD11b as the marker, 42 % for #6 and 46.24 % for #7, while that for luciferase knockdown control was 18.14% and parental line 4.97%. These initial data imply the involvement of HTm4 in myeloid lineage differentiation. Additionally, the knockdown of HTm4 delays the cell cycle entry into S phase. Since HTm4 is a hematopoietic cell specific cell cycle regulator, the cell cycle delay at G0/G1-S transition and higher differentiation efficiency observed in HTm4-knockdown WEHI-3 during myelocytic differentiation are likely related.