Array-based functional screening for genes that regulate vascular endothelial differentiation of Flk1-positive progenitors derived from embryonic stem cells.
Functional genomics is a central topic of current biological research, where a reverse genetic approach is one of the most promising strategies to discover functions of novel genes. Such an approach requires high-throughput methodologies to assess biological functions for a huge number of genes. We have developed a transfection array that permits parallel introduction of multiple plasmids separately into living cells. The feasibility of this array was examined in an assay system. Eleven genes were over-expressed alone, or in combination in vascular progenitors derived from embryonic stem cells. Endothelial differentiation of the cells was monitored through a stably transformed EGFP reporter construct that is expressed only in endothelial cells. Transcriptional activators that promote endothelial differentiation, such as Ets1 and Sox7, were identified. In addition, the assays also revealed an inhibitory effect on endothelial differentiation by several of the factors. These results demonstrate the feasibility of the transfection array for use in cell-based, high-throughput functional assays.
Yamauchi
Fumio
F
Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Okada
Mitsuhiro
M
Kato
Koichi
K
Jakt
Lars Martin
LM
Iwata
Hiroo
H
D000818:Animals; D001681:Biological Assay; D002454:Cell Differentiation; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000502:physiology*; D006609:High Mobility Group Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010957:Plasmids; D050784:Proto-Oncogene Protein c-ets-1 / Q000378:metabolism; D055760:SOXF Transcription Factors; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; C494042:Ets1 protein, mouse; D006609:High Mobility Group Proteins; D050784:Proto-Oncogene Protein c-ets-1; D055760:SOXF Transcription Factors; C102515:Sox7 protein, mouse; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases; D040301:Vascular Endothelial Growth Factor Receptor-2