| 摘要 |
A method and composition are disclosed for transfecting eukaryotic cells using a nucleic acid segment coupled to a site-specific chromosome-binding poylpeptide. The polypeptide-nucleic acid molecular conjugate preferably includes a recombinant DNA (rDNA) sequence, and is referred to herein as a polypeptide-linked-rDNA (PLR molecule). One example of a PLR molecule comprises an rDNA segment containing (a) a nucleotide sequence from a normally functioning structural gene, and (b) a selectable marker gene coding for antibiotic resitance, coupled by means of a covalent crosslinking reagent to a site-specific chromosome-binding polypetide (such as a transcription regulating polypeptide that binds to a specfic site in chromosomal DNA). After the PLR molecule enters a cell, by means such as electroporation, biolistics, etc., the chromosome-binding polypeptide enables transport of the PLR molecule through the cytosol and into the nucleus, using a nuclear localization sequence (NLS) domain of the polypeptide, or into a mitochondrion, using a mitochondrial leader peptide sequence. Inside the nucleus or mitochondrion, the polypeptide scans the chromosomes until it binds to a specific chomosomal binding site. This places the rDNA segment of the PLR molecule near a target site (such as an abnormal gene) in the chromosome. The desired rDNA segment contained in the PLR molecule has nucleotide sequence homology with the target gene, to enable cellular recombination enzymes to replace the targeted gene sequence with the desired gene sequence.
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