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Abstract
The production of animals with large transgenes is an
increasingly valuable tool in biotechnology and for
genetic studies, including the characterization and
manipulation of large genes and polygenic traits. In the
present study, we describe an intracytoplasmic sperm
injection (ICSI) method for the stable incorporation and
phenotypic expression of large Yeast Artificial
Chromosomes (YAC) constructs of submegabase and megabase
magnitude. By co-injecting spermatozoa and YACs into
metaphase II oocytes, we were able to produce founders
exhibiting germline transmission of an intact and
functional transgene of 250 kb, carrying the mouse
tyrosinase locus, used here as a reporter gene to rescue
the albinism of recipient mice. More than 35 %
transgenicity was obtained for this YAC-transgene. When
compared with the pronuclear microinjection standard
method, the efficiency of the ICSI-Mediated YAC Transfer
system was significantly greater. In summary, we describe
for the first time, stable incorporation in the host
genome and correct phenotypic expression of large DNA
constructs mediated by ICSI.
Key words:
Gamete Biology •
Assisted Reproductive Technology •
Early development •
Gene regulation •
In vitro fertilization
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