Introduction. ALS is a fatal neurodegenerative disease that
occurs in two forms: sporadic and familial, the latter linked to
mutations in the SOD1 gene. Pathogenic hypotheses mainly rely
on transgenic rodent models however doubts have been raised
about their suitability to faithfully reproduce the human disease.
Therefore, a more suitable model is strongly demanded to provide
a better tool to study the disease and to facilitate the preclinical
findings extrapolation. Swine model plays an emerging
role in biomedical research as its anatomical, physiological and
biochemical features are more closely related to human species.
On this basis, our group produced, by Genetic Engineering and
SCNT, transgenic swine blastocysts carrying the hSOD1G93A
mutation, which is the most frequently studied in rodents, since
it reproduces the ALS patients phenotype progression.
Material and Methods. Using the Multisite Gateway System
(Invitrogen) we obtained the “EntryClone” pENTRL1L2-
hSODG93A, containing the cDNA coding the hSOD1G93A
gene whose open reading frame was confirmed by sequencing,
and the “DestinationVector” pMGOrfA5¢3’MARpuro5171.
The exchange reaction between the “DestinationVector” and
the “EntryClone” was mediated by LR Clonase-Invitrogen,
and was used to transform chemically competent E.coli cells
(OneShotMatch1-Invitrogen). The resulting “ExpressionVector”
pMG5¢3’MARPuro-hSODG93A was purified and analyzed.
The same construct was successfully used to develop an ubiquitous
EGFP expression vector that maintains high expression
level through the next generation of pigs. This vector carries
the pCAGGS hybrid promoter (CMV-IE enhancer + chicken
β-actin promoter) inserted between two insulators (5′MAR of
chicken lysozyme gene) to prevent positional or copy number
silencing effects. 5 µg of linearized vector were used to transfect
1 × 106
Pig Adult Fibroblast (PAF) cultured in DMEM/
M199[1:1] + 10%FCS+1% β-FGF by Nucleofector (Amaxa
Biosystem). Transgene expression was detected by ICC using an
anti-hSOD1 antibody ([1:200]07–403 Millipore) to select PAF
colonies to be employed as donor cells. The endogenous expression
level was evaluated in Huvec cells. SCNT-embryos were
reconstructed following a zona-free method, as described previously.
Transgenic hSOD1G93A-embryos were finally grown in
vitro to assess morphology, development and survival.
Results. PAFs showed a transgene expression level ranging
from lower (about 0.5 times) to higher (about 3–4 times) than
the endogenous one. Thirteen hSOD1G93A-PAF colonies carrying
the complete range of expression levels were employed in
eight SCNT experiments. A total of 1,630 oocytes were enucleated
and 1430 SCNT-embryos were obtained. The average
viable embryos percentage was 39.16% of the total manipulated
oocytes. These results, consistent with those obtained from similar
experiments conducted with other transgenes, have ruled out
the hSOD1G93A lethality in the pre-implantation embryonic
phases, regardless of its expression level.
Conclusion. On the basis of these results the hSOD1G93Ablastocysts
can be generated by SCNT. The next step will be
to implant them in recipient sows to generate offspring. Taking
into account the similarity in size and physiology of neuromuscolar
system, a swine ALS model may represent a more suitable
model in reproducing the human disease than current transgenic
|Numero di pagine||1|
|Stato di pubblicazione||Pubblicato - 2012|
|Evento||PRION - International Prion Congress - Amsterdam, The Netherlands|
Durata: 9 mag 2012 → 12 mag 2012
- Cu/Zn superoxide-dismutase1 (SOD1)
- amyotrophic lateral sclerosis (ALS)
- somatic cell nuclear transfer (SCNT)
- swine model