Gene targeting for somatic cell manipulation: rapid analysis of reduced chromosome hybrids by Alu-PCR fingerprinting and chromosome painting.
The techniques of reverse genetics rely heavily on parasexual methods for manipulating the human genome. However, the application of somatic cell genetics is severely limited by the availability of suitable endogenous selectable markers in the genome. We have addressed this problem by targeting a universally selectable marker into a predetermined region of the genome, using a stringent selection for homologous recombination. Correct gene targeting to human chromosome 7q11 was screened for by Southern blotting and confirmed by fluorescent in situ hybridization. Reduced chromosome 7 hybrids were generated by chromosome mediated gene transfer and selection for the neo gene. The resultant transgenomes were characterized by a combination of L1 fingerprinting, locus specific marker analysis, Alu-PCR and chromosome 'painting'. Alu-PCR and L1 'fingerprints' are complementary and mutually consistent. Chromosome 'painting' reflects and extends the results obtained for specific marker co-transfer. Thus Alu-PCR 'fingerprinting' and 'painting' combine to rapidly provide an accurate picture of transgenome content and complexity. Gene targeting, chromosome tagging and subsequent isolation can be applied to any region of the genome for which a molecular probe is available.
Human Genetics Unit, Western General Hospital, Edinburgh, UK.
J R
JR
Dorin
E
E
Emslie
D
D
Hanratty
M
M
Farrall
J
J
Gosden
D J
DJ
Porteous
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002871:Chromosome Banding; D002874:Chromosome Mapping / Q000379:methods*; D002897:Chromosomes, Human, Pair 7; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D016172:DNA Fingerprinting / Q000379:methods; D015342:DNA Probes; D015252:Deoxyribonucleases, Type II Site-Specific; D005801:Genes, Homeobox; D015894:Genome, Human; D006801:Humans; D017403:In Situ Hybridization; D019868:Kanamycin Kinase; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010770:Phosphotransferases / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics
D015342:DNA Probes; D015345:Oligonucleotide Probes; D004247:DNA; D010770:Phosphotransferases; D019868:Kanamycin Kinase; C024669:endodeoxyribonuclease AluI; D015252:Deoxyribonucleases, Type II Site-Specific