Stable gene expression from a mammalian artificial chromosome.
We have investigated the potential of PAC-based vectors as a route to the incorporation of a gene in a mammalian artificial chromosome (MAC). Previously we demonstrated that a PAC (PAC7c5) containing alpha-satellite DNA generated mitotically stable MACs in human cells. To determine whether a functional HPRT gene could be assembled in a MAC, PAC7c5 was co-transfected with a second PAC containing a 140 kb human HPRT gene into HPRT-deficient HT1080 cells. Lines were isolated containing a MAC hybridizing with both alpha-satellite and HPRT probes. The MACs segregated efficiently, associated with kinetochore proteins and stably expressed HPRT message after 60 days without selection. Complementation of the parental HPRT deficiency was confirmed phenotypically by growth on HAT selection. These results suggest that MACs could be further developed for delivering a range of genomic copies of genes into cells and that stable transgene expression can be achieved.
Grimes
B R
BR
MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.
Schindelhauer
D
D
McGill
N I
NI
Ross
A
A
Ebersole
T A
TA
Cooke
H J
HJ
D015152:Blotting, Northern; D002455:Cell Division; D002460:Cell Line; D022201:Chromosomes, Artificial; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D005821:Genetic Techniques; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence; D018895:Microsatellite Repeats; D008856:Microscopy, Fluorescence; D008938:Mitosis; D010641:Phenotype; D013997:Time Factors; D014162:Transfection; D019076:Transgenes
D018076:DNA, Complementary; D007041:Hypoxanthine Phosphoribosyltransferase