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In JoVE (1)
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Articles by Guang Hu in JoVE
JoVE General
Oct4GiP Assay כתב ללמוד גנים המווסתים גזע עובריים עכבר נייד תחזוקה עצמית והתחדשות
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences
אנו מתארים assay כתב הקרינה במהירות לזהות ולאפיין את הגנים המווסתים גזע עובריים של עכברים תא תחזוקה עצמית והתחדשות.
Other articles by Guang Hu on PubMed
[The Distribution and Origin of P Elements in Drosophila Melanogaster Strains from Northeast China]
Eukaryotic transposable elements, especially P transposable elements of Drosophla, are very important to study the evolution of biology. We collected 130 single--female lines of D. melanogaster from 13 places in Northeast China and 3 places nearby which are Beijing(BC), Yantai (YT), Huhehaote(HHT) in 1999 and 2000. We amplified the fragments from ORF2 to ORF3 of P elements using PCR and calculated the defective frequencies and defective parameters of P elements from different places. Basing on the distribution of defective P elements, we suggested the invasion route of P elements of D. melanogaster in Northeast China. The results indicate that the defective frequencies decrease from the frontier to the inland, and in the isolated places the frequencies are lower. It is proposed that P elements of D. melanogaster in Northeast China originate from Korea and Russia to the frontiers of P.R. China, then expand into the inland.
A Critical Role for Calponin 2 in Vascular Development
Calponin 2 (h2 calponin, CNN2) is an actin-binding protein implicated in cytoskeletal organization. We have found that the expression of calponin 2 is relatively restricted to vasculature from 16 to 30 h post-fertilization during zebrafish (Danio rerio) development. Forty-eight hours after injecting antisense morpholino oligos against calponin 2 into embryos at the 1-4-cell stage, zebrafish demonstrated various cardiovascular defects, including sluggish axial and head circulation, absence of circulation in intersegmental vessels and in the dorsal longitudinal anastomotic vessel, enlarged cerebral ventricles, and pericardial edema, in addition to an excess bending, spiraling tail and twisting of the caudal fin. Knockdown of calponin 2 in the Tg(fli1:EGFP)(y1) zebrafish line (in which a fli1 promoter drives vascular-specific enhanced green fluorescent protein expression) indicated that diminished calponin 2 expression blocked the proper migration of endothelial cells during formation of intersegmental vessels. In vitro studies showed that basic fibroblast growth factor-induced human umbilical vein endothelial cell migration was down-regulated by knockdown of calponin 2 expression using an antisense adenovirus, and overexpression of calponin 2 enhanced migration and hastened wound healing. These events were correlated with activation of mitogen-activated protein kinase; moreover, inhibition of this pathway blocked the promigratory effect of calponin 2. Collectively, these data suggest that calponin 2 plays an important role in the migration of endothelial cells both in vivo and in vitro and that its expression is critical for proper vascular development.
A Novel Endothelial-specific Heat Shock Protein HspA12B is Required in Both Zebrafish Development and Endothelial Functions in Vitro
A zebrafish transcript dubbed GA2692 was initially identified via a whole-mount in situ hybridization screen for vessel specific transcripts. Its mRNA expression during embryonic development was detected in ventral hematopoietic and vasculogenic mesoderm and later throughout the vasculature up to 48 hours post fertilization. Morpholino-mediated knockdown of GA2692 in embryos resulted in multiple defects in vasculature, particularly, at sites undergoing active capillary sprouting: the intersegmental vessels, sub-intestinal vessels and the capillary sprouts of the pectoral fin vessel. During the course of these studies, a homology search indicated that GA2692 is the zebrafish orthologue of mammalian HspA12B, a distant member of the heat shock protein 70 (Hsp70) family. By a combination of northern blot and real-time PCR analysis, we showed that HspA12B is highly expressed in human endothelial cells in vitro. Knockdown of HspA12B by small interfering RNAs (siRNAs) in human umbilical vein endothelial cells blocked wound healing, migration and tube formation, whereas overexpression of HspA12B enhanced migration and accelerated wound healing - data that are consistent with the in vivo fish phenotype obtained in the morpholino-knockdown studies. Phosphorylation of Akt was consistently reduced by siRNAs against HspA12B. Overexpression of a constitutively active form of Akt rescued the inhibitory effects of knockdown of HspA12B on migration of human umbilical vein endothelial cells. Collectively, our data suggests that HspA12B is a highly endothelial-cell-specific distant member of the Hsp70 family and plays a significant role in endothelial cells during development and angiogenesis in vitro, partially attributable to modulation of Akt phosphorylation.
Forward RNAi Screens in Primary Human Hematopoietic Stem/progenitor Cells
The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation, we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex, pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion, respectively, thereby demonstrating the potential of this forward screening approach in primary human stem cell populations.
