Abstract
Vol.14 No.1
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Subcloning of Mouse A3-1 Embryonic Stem Cells
JMOR, 14(1) 41-44, 1997
DOI: 10.1274/jmor.14.41
DOI: 10.1274/jmor.14.41
Three methods for subcloning embryonic stem (ES) cells, namely the isolation, precipitation and colony pickup method, were examined as follows. Isolation method: ES cells were trypsinized and classified morphologically as spherical and unspherical cells. Each type of ES cell was isolated with a micropipette and micromanipulator and was cultured separately. Precipitation method: ES cells were trypsinized and incubated for 15-45 min. Both the adhesive and floating cells were cultured separately. Colony pickup method: morphologically undifferentiated ES cell colonies were picked up with a micropipette. The colony was trypsinized and replated on a new feeder layer. Before the subcloning, there were 30-48% of normal diploid sets of 40 chromosomes in ES cells. Both the isolation and precipitation methods have not shown the effect of subcloning on the karyotype. The most effective subcloning method in this study was the colony pickup method. All of three sublines derived by the colony pickup method showed 68-78% of normal karyotypes and simple embryoid and cystic embryoid bodies were formed 3-4 days and 7-8 days after suspension culture, respectively.
