Inducible expression of RANKL in transgenic pigs under the control of the Tet-On system

Because of the tremendous need for transgenic large animal models
for human diseases, the process of SCNT is a crucial step in
transgenic pig production. In our study, we evaluated the
particular steps during the production for their impact on the
efficiency of cloning transgenic pigs. For this purpose,
statistical analysis was performed for all SCNT data from the years
2006 until June 2010. The RANKL transgenic osteoporosis model was
chosen for an example for the production steps needed to finally
achieve a disease model, to elucidate pitfalls and chances of SCNT
procedure. In total 151 in vivo SCNT experiments using different
transgenic cell lines were carried out, resulting in 243 piglets
and fetuses. Statistical analysis revealed that donor cells treated
exclusively in our laboratory had a significant better birth rate
than donor cell originated of other laboratories. Furthermore,
there was a significant relation between number of transferred NT
embryos and later pregnancy checks, birth rate and abortion rate.
The more NT embryos were transferred, the more pregnancies finished
to terms. It was also elucidated that in our studies a different in
vitro culture time of 24 or 48 hours had no significant impact on
the outcome like pregnancy or birth rate. Seasonal changes during
the years had no significant influence on pregnancy rate, birth or
abortion. But there was a strong tendency that autumn showed best
performance of all seasons, and most pregnancies were lost after
embryo transfers during the summer. All these findings will be
integrated in future in vivo SCNT experiments and embryo transfers.
For the production of a transgenic osteoporosis model 17 in vivo
experiments took place so far, with an outcome of 4 fetuses and 25
piglets. For gaining a controllable expression of RANKL, it was
necessary to establish double transgenic pigs to sidestep harmful
effects of RANKL overexpression during the fetal development. First
attempts to integrate both genes, tetracycline controlled
transactivator (Tet-On) and RANKL, in a single step of cell
transfection and SCNT, had no satisfying result. We obtained 4
fetuses and stillborn recloned piglets carrying both genes, but
they showed only expression of Tet-On and it was impossible to
induce RANKL overexpression. Therefore the strategy was changed in
favor to two rounds of transfection and nuclear transfer. First
Tet-On transgenic piglets were established and screened for
integration and expression. Piglet 9894 showed the best expression
and severed as donor for next cell transfection step. These Tet-On
+ TARE RANKL cells were in vitro tested for their inducibility.
Thereafter SCNT and embryo transfer of the best candidate were
performed and they resulted in 4 pregnancies which all finished to
term. One double transgenic piglet could be raised and will be kept
until adulthood to establish a line of Tet-On +TARE RANKL
transgenic pigs. Importantly, this founder animal showed inducible
RANKL overexpression. Other constructs might be based on the
existing Tet-On cell line in the future, offering an inducible
system for a broad variety of different transgenes. Thus a
functional Tet-On system in the pig is reported for the first time.