Easy assessment of ES cell clone potency for chimeric development and germ-line competency by an optimized aggregation method

Production of germ-line competent chimeric mice from embryonic stem (ES) cells is an inevitable step in establishing gene-manipulated mouse lineages. A common method used for creating chimeric mice is the injection of ES cells into the blastocoelic cavity (blastocyst injection). The aggregation meth...

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Bibliographic Details
Published in:Journal of biochemical and biophysical methods 1999-05, Vol.39 (3), p.137-142
Main Authors: Kondoh, Gen, Yamamoto, Yoichi, Yoshida, Kayo, Suzuki, Yutaka, Osuka, Soh, Nakano, Yuka, Morita, Takashi, Takeda, Junji
Format: Article
Language:English
Subjects:
Aggregation
Animals
Chimera - physiology
Chimerism
Coculture Techniques
Embryo, Mammalian - physiology
Embryonic stem cell
Female
Genetic Engineering - methods
Germ Cells
Germ-line transmission
Mice
Mice, Inbred C57BL
Mice, Inbred Strains
Mutation - physiology
Stem Cells - physiology
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Summary:Production of germ-line competent chimeric mice from embryonic stem (ES) cells is an inevitable step in establishing gene-manipulated mouse lineages. A common method used for creating chimeric mice is the injection of ES cells into the blastocoelic cavity (blastocyst injection). The aggregation method is an alternative way to introduce ES cells to the host embryo which is less difficult than blastocyst injection. Here we re-examined the condition of embryo-ES cell coculture on the aggregation method and found improvement of germ-line competent chimeric production by a simple modification of the coculture medium. Moreover, R1 ES cell and its 10 gene-manipulated subclones were tested by this method. Although all ES cell clones showed good morphology and a normal karyotype, the efficiency of chimeric development and germ-line transmission varied among clones and were classified into three grades according to germ-line competency. In the first group (class A), both the incidence of chimera with high ES cell contribution and the rate of germ-line transmission were fairly high. Germ-line competent chimeras were obtained but with rather low efficiency in the second group (class B), while another group (class C) showed an absence of high ES cell-contributed chimeras and no germ-line transmission. These results suggest the usefulness of this modified aggregation method to predict the potency of ES cell clones for germ-line competency.
ISSN:0165-022X
1872-857X
DOI:10.1016/S0165-022X(99)00008-1