Bacterial transfer of large functional genomic DNA into human cells.
Efficient transfer of chromosome-based vectors into mammalian cells is difficult, mostly due to their large size. Using a genetically engineered invasive Escherichia coli vector, alpha satellite DNA cloned in P1-based artificial chromosome was stably delivered into the HT1080 cell line and efficiently generated human artificial chromosomes de novo. Similarly, a large genomic cystic fibrosis transmembrane conductance regulator (CFTR) construct of 160 kb containing a portion of the CFTR gene was stably propagated in the bacterial vector and transferred into HT1080 cells where it was transcribed, and correctly spliced, indicating transfer of an intact and functional locus of at least 80 kb. These results demonstrate that bacteria allow the cloning, propagation and transfer of large intact and functional genomic DNA fragments and their subsequent direct delivery into cells for functional analysis. Such an approach opens new perspectives for gene therapy.
Laner
A
A
Department of Medical Genetics, Childrens Hospital, Ludwig Maximilians University, Munich, Germany.
Goussard
S
S
Ramalho
A S
AS
Schwarz
T
T
Amaral
M D
MD
Courvalin
P
P
Schindelhauer
D
D
Grillot-Courvalin
C
C
D045744:Cell Line, Tumor / Q000378:metabolism / Q000382:microbiology*; D022202:Chromosomes, Artificial, Bacterial; D022222:Chromosomes, Artificial, Human; D002999:Clone Cells; D004274:DNA, Recombinant / Q000378:metabolism*; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics*; D005434:Flow Cytometry; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage*; D016680:Genome, Bacterial; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012509:Sarcoma
D004274:DNA, Recombinant