By Tenneille Ludwig, James A. Thomson (auth.), John R. Masters, Bernhard O. Palsson, James A. Thomson (eds.)
If you should develop or signify embryonic stem cells or convince them to distinguish right into a specific mobilephone kind, then this booklet comprises info that's very important in your luck. the purpose is to supply transparent uncomplicated directions and protocols for turning out to be, retaining and characterizing embryonic stem cells and info of many of the tools used to make stem cells differentiate into particular mobile varieties. The contents should be of curiosity to stem phone biologists, tissue engineers and scientists wishing to take advantage of embryonic stem cells for healing reasons. every one bankruptcy has been written and edited via the world over revered scientists operating on the leading edge of technological advancements in human embryonic stem cells.
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Extra resources for Human Cell Culture: Volume VI: Embryonic Stem Cells
Example text
R. ), Embryonic Stem Cells, 41–52. © 2007 Springer. qxd 18/4/07 4:45 PM Page 42 42 Zwaka 2. EXOGENOUS GENE EXPRESSION IN HUMAN ES CELLS Genes have been introduced into human ES cells by both transfection and infection techniques. Transient transfection, the simplest method for introducing new genes into various cell types, may be achieved using liposomes and other cationic/ lipid-based particles, brief electric shocks (electroporation), or other strategies. However, the expression of transiently transfected DNA tends to be poorly controlled and the DNA tends to disappear within days or weeks, or in rare cases may integrate randomly into the host DNA.
2004 and unpublished observation). Typically, when DNA is introduced by transient transfection, it is transferred in the form of a circular plasmid (Weintraub et al. 1986). Under these conditions transcription is initiated within a few hours after transfection and maximal transgenic protein expression is observed within 1–3 days. The number of plasmids taken up by each cell may vary from a single plasmid to hundreds or more. Consequently, expression levels may vary dramatically among individual cells, complicating the interpretation of some transient overexpression experiments.
After noggin treatment, colonies were cut into small pieces using a 27 gauge needle and pieces transferred to individual wells in a non-adherent 96-well plate to allow neurosphere formation. Neurospheres were cultured in suspension in neural basal medium (NBM) with supplements, as described by Reubinoff and colleagues (Reubinoff et al. 2001), for 1–2 weeks. Subsequent differentiation was carried out by plating the spheres onto laminin or fibronectin for adherent culture under conditions previously described for neural or glial differentiation respectively (Reubinoff et al.