Labeling embryonic stem cells with enhanced green fluorescent protein on the hypoxanthineguanine phosphoribosyl transferase locus.
OBJECTIVE
To label embryonic stem (ES) cells with enhanced green fluorescent protein (EGFP) on the hypoxanthineguanine phosphoribosyl transferase (HPRT) gene locus for the first time to provide a convenient and efficient way for cell tracking and manipulation in the studies of transplantation and stem cell therapy.


METHODS
Homologous fragments were obtained by polymerase chain reaction (PCR), from which the gene targeting vector pHPRT-EGFP was constructed. The linearized vector was introduced into ES cells by electroporation. The G418(r)6TG(r) cell clones were obtained after selection with G418 and 6TG media. The integration patterns of these resistant cell clones were identified with Southern blotting.


RESULTS
EGFP expressing ES cells on the locus of HPRT were successfully generated. They have normal properties, such as karyotype, viability and differentiation ability. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages and in differentiated cells. Cultured in suspension, the "green" ES cells aggregated and formed embryoid bodies, retaining the green fluorescence at varying developmental stages. The "green" embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence.


CONCLUSIONS
This generation of "green" targeted ES cells is described in an efficient protocol for obtaining the homologous fragments by PCR. Introducing the marker gene in the genome of ES cells, we should be able to manipulate them in vitro and use them as vehicles in cell-replacement therapy as well as for other biomedical and research purposes.
Teng
Lu
L
Laboratory of Genetics, College of Life Science, Peking University, Beijing 100871, China.
Meng
Guoliang
G
Xing
Yang
Y
Shang
Kegang
K
Wang
Xiaoke
X
Gu
Jun
J
D000818:Animals; D002478:Cells, Cultured; D002874:Chromosome Mapping; D004622:Embryo, Mammalian / Q000166:cytology*; D049452:Green Fluorescent Proteins; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D011995:Recombination, Genetic; D013234:Stem Cells / Q000378:metabolism*; D019076:Transgenes
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D007041:Hypoxanthine Phosphoribosyltransferase