Mechanism of chromosomal integration of transgenes in microinjected mouse eggs: sequence analysis of genome-transgene and transgene-transgene junctions at two loci.
Production of transgenic animals is a key technique in modern biology, but the process of chromosomal integration of transgenes in microinjected eggs is still not fully understood. To gain information on the mechanisms involved in this process, we have cloned two transgene loci and their corresponding pre-integration sites and compared the junction sequences with the parental nucleotide (nt) sequences. No extensive DNA rearrangements were detected at these loci: only simple deletions (caused by the integration of the transgene concatemers) were present in the host genome. Analysis of three transgene-transgene junctions within the concatemers showed that 'nibbling' of ends (up to 3 nt) had occurred at some ends prior to joining. At all four genome-transgene junctions, short homologies of 1 to 3 nt were found, and at least three of these junctions were associated with the consensus sequence for topoisomerase-I cleavage sites. Moreover, three of the four integration junctions occurred in the terminal regions of the injected sequence, at positions only a few nt away from the ends. These results suggest that linear, but not circular, concatemers were preferentially integrated at their ends utilizing short homologies to the host genome.
Hamada
T
T
Research Laboratory for Genetic Information, Kyushu University Fukuoka, Japan.
Sasaki
H
H
Seki
R
R
Sakaki
Y
Y
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004264:DNA Topoisomerases, Type I; D004251:DNA Transposable Elements; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D004264:DNA Topoisomerases, Type I