A LacZ-based transgenic mouse for detection of somatic gene repair events in vivo.

Somatic gene repair of disease-causing chromosomal mutations is a novel approach for gene therapy. This method would ensure that the corrected gene is regulated by its endogenous promoter and expressed at physiological levels in the appropriate cell types. A reporter mouse, Gtrosa26(tm1Col), was generated by targeting a mutated LacZ gene to the Rosa26 locus in mouse embryonic stem (ES) cells. The LacZ gene contains a G to A point mutation, resulting in a Glu to Lys amino-acid substitution at position 461, which abrogates enzymatic activity. The gene is expressed in ES cells, primary embryonic fibroblasts, and in all tissues examined in the adult mouse, including the lung, liver, kidney, spleen, heart, brain and smooth muscle. This transgenic mouse will allow testing of gene repair strategies in vivo and identification of which cell types can be successfully targeted by chromosomal gene repair. Although low levels of gene repair were achieved in the ES cells used to generate the Gtrosa26(tm1Col) mouse, preliminary attempts at gene repair in vivo were unsuccessful, thus highlighting the difficulties that will have to be overcome to get this approach to work.

A comparison of gene repair strategies in cell culture using a lacZ reporter system.

Synthetic oligonucleotides and DNA fragments of less than 1 kilobase (kb) have been shown to cause site-specific genetic alterations in mammalian cells in culture and in vivo. We have used a lacZ reporter gene system to compare the efficiency of episomal and chromosomal gene repair in human embryonic kidney epithelial cells (HEK293), Chinese Hamster Ovary fibroblasts (CHOK1), human bronchial epithelial cells (16HBE), and mouse embryonic stem (ES) cells. The lacZ gene contains a G to A nucleotide change, (Glu to Lys mutation) that abrogates beta-galactosidase activity. We compared the efficiency of different gene repair methods to correct this mutation and restore beta-galactosidase activity. We evaluated PCR-generated double-stranded DNA fragments of 0.52-1.9 kb, single-stranded DNA oligonucleotides of 20, 35, or 80 bases containing internal phosphorothioate links, and a 68 base RNA:DNA oligonucleotide. All of the oligonucleotides and DNA fragments showed some gene repair ability with an episomal plasmid. Short DNA fragments of 0.52 kb or greater gave the highest frequencies of episomal gene repair while single-stranded DNA oligonucleotides gave the highest frequency of chromosomal repair. In the context of a chromosomal target, antisense DNA oligonucleotides gave 5-fold higher frequencies of gene repair than their sense counterparts. The RNA:DNA chimeric oligonucleotide gave little or no gene repair on either a chromosomal or episomal target.