Integration of exogenous DNA into mouse embryonic stem cell chromosomes shows preference into genes and frequent modification at junctions.

Chromosomal integration of exogenous DNA in mammalian cells allows stable gene expression for a variety of biological applications. Although it is presumably mediated by DNA repair machinery, little is known regarding site preferences and other characteristics. We isolated and analyzed 256 chromosomal-plasmid DNA integration junctions from 158 plasmid integrants after electroporation in mouse embryonic stem (ES) cells.

Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector.

Background: Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR.

Methods: We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells.

Correction of chromosomal mutation and random integration in embryonic stem cells with helper-dependent adenoviral vectors.

For gene therapy of inherited diseases, targeted integration/gene repair through homologous recombination (HR) between exogenous and chromosomal DNA would be an ideal strategy to avoid potentially serious problems of random integration such as cellular transformation and gene silencing. Efficient sequence-specific modification of chromosomes by HR would also advance both biological studies and therapeutic applications of a variety of stem cells. Toward these goals, we developed an improved strategy of adenoviral vector (AdV)-mediated HR and examined its ability to correct an insertional mutation in the hypoxanthine phosphoribosyl transferase (Hprt) locus in male mouse ES cells.

Detachment analysis of the translocated W chromosome shows that the female-specific randomly amplified polymorphic DNA (RAPD) marker, female-218, is derived from the second chromosome fragment region of the translocated W chromosome of the sex-limited p(B) silkworm (Bombyx mori ) strain.

The sex chromosomes of the silkworm, Bombyx mori, are designated ZW for the female and ZZ for the male. We previously characterized a female-specific randomly amplified polymorphic DNA (RAPD) marker, designated Female-218, from the translocation-bearing W chromosomes. These W chromosomes contain a region of the second chromosome, which carries visible larval markers of the p loci.