Using the mouse model, intracytoplasmic sperm injection (ICSI)-mediated transgenesis has been
shown to be a valuable tool for the production of transgenic animals, an essential instrument for basic and
applied research in bioscience. This method of transgenesis consists of the microinjection of spermatozoa preincubated
with foreign DNA. ICSI of DNA-loaded sperm cells has been shown to mediate mouse
transgenesis at high efficiency, especially when sperm cell damage (by freeze-thaw cycles or exposure to
detergents) is induced. The greatest advantage of ICSI-mediated transgenesis is that it allows introduction of
very large DNA transgenes (e.g., yeast artificial chromosomes), with relatively high efficiency into the
genome of hosts, as compared to pronuclear microinjection. In addition, ICSI-mediated transgenesis is
associated with (a) low frequencies of embryo mosaicism, one of the major limitations of pronuclear
microinjection for the production of transgenic livestock, and (b) a high frequency of Mendelian germline
transmission of transgenic sequences among founder animals. In this chapter we will review some factors that
can increase the efficiency of the ICSI-mediated transgenesis and we will describe a new active form of ICSImediated
transgenesis employing fresh sperm in conjunction with recombinase or transposase molecules.
Keywords: Intracytoplasmic sperm injection, Mice, Large DNA transgenes, Efficiency, Sperm cell damage,
Active transgenesis, Rec A, Mosaicism, DNA integration, Matrix attachment regions, Transmission.
