Inadvertent transgenesis by conventional ICSI in mice.
BACKGROUND
ICSI is a relatively new treatment for human male-related infertility, as well as an efficient method for the production of transgenic animals by injecting into the oocyte sperm previously incubated with foreign DNA. As semen samples collected in human infertility clinics are frequently contaminated with bacteria, one risk associated with the ICSI procedure is the injection of foreign, sperm-associated exogenous DNA into the oocyte, and the generation of transgenic offspring.


METHODS
To analyse this possibility, ICSI was performed in mouse oocytes with frozen-thawed and Percoll-treated fresh sperm samples intentionally contaminated with plasmid EGFP-transformed E. coli bacteria or medium from which these bacteria were washed. Fertilized embryos were cultured in vitro until morula/blastocyst stage, transferred into pseudopregnant females, and at day 14, fetuses and reabsorptions were analysed by PCR for the genomic presence of integrated plasmid and/or bacterial DNA.


RESULTS
Independently of the sperm pretreatment tested, transgenesis was produced.


CONCLUSIONS
Inadvertent transgenesis by conventional ICSI is a possibility that should not be neglected. Particular precautions, such as full bacteriological semen examinations and effective antibiotic semen pretreatments, should be taken in human infertility clinics, in order to exclude the possibility of accidental transgenesis.
Moreira
Pedro Nuno
PN
Departamento de Reproducción Animal, INIA, Ctra. de la Coruña Km. 5.9 Madrid 28040, Spain. pmoreira@inia.es
Fernández-González
Raúl
R
Rizos
Dimitrios
D
Ramirez
Miguel
M
Perez-Crespo
Miriam
M
Gutiérrez-Adán
Alfonso
A
D000818:Animals; D001755:Blastocyst / Q000382:microbiology; D019457:Chromosome Breakage / Q000235:genetics; D002875:Chromosomes / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000382:microbiology*; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro; D005615:Freezing; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007202:Indicators and Reagents / Q000494:pharmacology; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009865:Oocytes / Q000378:metabolism; D009924:Organ Culture Techniques; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020554:Sperm Injections, Intracytoplasmic / Q000009:adverse effects*; D019076:Transgenes / Q000235:genetics*
D007202:Indicators and Reagents; D049452:Green Fluorescent Proteins; D004247:DNA