1. Introduction
Microinjection of foreign DNA into pronuclei of fertilized oocytes has been the only Ž
. successful method for the generation of transgenic livestock Table 1 . Although this
Ž procedure works reliably, it is inefficient 1–4 transgenic offspringrtransferred mi-
. croinjected zygotes , results in random integration into the host genome and variable
Ž .
expression due to position effects Pursel and Rexroad, 1993; Wall, 1996 . In addition, it is time consuming and requires substantial intellectual, financial and material resources
Ž .
Seidel, 1993 . As a consequence, research has focused on alternate methodologies for improving the generation of transgenic livestock. These include sperm mediated DNA
Ž .
Ž .
transfer Gandolfi, 1998; Squires, 1999 , the intracytoplasmic injection ICSI of trans- Ž
. genic sperm heads Perry et al., 1999 , the use of retroviral vectors either by injection or
Ž .
infection of oocytes or embryos Haskell and Bowen, 1995; Chan et al., 1998 or the use Ž
of genetically modified ovine, bovine or caprine donor cells in nuclear transfer Schnieke .
et al., 1997; Cibelli et al., 1998a; Baguisi et al., 1999 . Further improvements may be derived from the adaptation of technologies that are effective in allowing precise
genomic modifications of the murine genome. These include targeted chromosomal integration by DNA recombinases, such as Cre or FLP or homologous recombination
that would enable to generate transgenic animals with a gain or a loss of function
Table 1 Methodological repertoire for the production of genetically modified mice and large farm animals
q sshown; y s not shown; ?s questionable; approaches were validated according to germline transmission and expression data.
Methodology Integration
Expression Mouse
Livestock Gain-of-function
Microinjection minigene constructs
random variable
q q
Ž .
with regulatable promoter random
inducible MtrTET qrq
qry artificial chromosomes
random integration site independent
q y
Ž .
YAC, BAC Retroviral infection
randomrsite variable?
q q?
specific Ž
. Sperm atogonia mediated
random ?
q q?
Targeted chromosomal integration defined
controlled q
y Ž
. Flp, Crerlox
a
Nuclear transfer with transgenic randomrdefined
variabler? y
qrq donor cells
Loss-of-function ES cellsqgene knockout
defined abolished in all cells
q y
Cell-type-specific knockout defined
abolished in specific cells q
y Inducible knockout
defined abolished upon induction
q y
Somatic nuclear transfer with defined
abolished y?
y? knockout cells
a
Ž .
Pharmaceutical Proteins Limited PPL has recently announced the birth of transgenic lambs produced by the gene targeting method and nuclear transfer.
Ž . Ž
. knockout
Capecchi, 1989; Kilby et al., 1993 . In light of the recent advances, somatic nuclear transfer holds the greatest promise for significant improvements in the genera-
tion of transgenic livestock. A major prerequisite is the availability of suitable cell lines compatible with techniques for precise genetic modifications either for gain or loss of
function. Another premise is a significantly improved knowledge of gene sequences and organization of the livestock genome, which currently is lagging much behind that of
mouse and human, where the putative 3 billion bp are expected to be sequenced by the year 2003. This review focuses on recent achievements in transgenic livestock as
generated via microinjection and briefly outlines the potential for improving transgenic technology in livestock species by nuclear transfer and application of sophisticated
molecular tools.
2. Current technology — application of microinjection
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