uptake by sperm when electroporation or lipofection is used. However, during
nanoSMGT, the ratios of linear-to-circular plasmid did not influence the uptake
by sperm cells and none of the tested treatments affected sperm motility and
viability after nanotransfection [130, 131]. Campos et al. [131] also used halloysite
clay nanotubes (HCN) for nanoSMGT. They observed that the mean number of
plasmids taken up by cattle sperm was higher in HCN-based gene delivery than by
using lipofection. IVF of oocytes with HCN-transfected sperm successfully resulted
in transgenic embryos with higher efficiency but, unfortunately, the transgene did
not express.
5.2 NP-Based Gene Delivery into Oocytes
Transgenic animal production via introduction of genes into oocytes have not been
very fruitful by conventional methods due to the high cytoplasmic content of
nuclease enzymes, cytoplasmic sequestration of injected DNA by DNA binding
proteins and cytoskeletal elements and the lack of DNA to transport across the
nuclear membrane. Some studies have shown that simultaneous introduction of
DNA along with the sperm can lead to successful production of transgenic mice
[132]. However, this method, called MII transgenesis, required intracytoplasmic
sperm injection to be performed and was not very fruitful in non-rodent species.
Use of lentriviral vectors, on the other hand, has resulted in successful production of
transgenic animals via gene introduction in oocytes.
MII-stage oocytes lack a nuclear membrane and offer a unique opportunity for
DNA to interact with the host chromatin to produce transgenic animals. However,
all efforts to produce transgenic animals via direct injection of DNA into oocytes
have failed. We have recently developed a NP-based gene delivery method for
mammalian oocytes, called oocyte-mediated gene transfer (OMGT) (Fig. 4). Pig
oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured
in vitro for 42–44 h and microinjected with DNA NP solution (10 ng/mL) using a
femtojet microinjector (Eppendorf, Hamburg, Germany). The DNA (4.7 kb) was
derived from the pEGFP-C1 plasmid (Clontech Laboratories, CA, USA), which
contains EGFP-encoding transgene under the control of cytomegalovirus (CMV)
promoter, and linearized with ApaLI restriction enzyme. Injected oocytes were then
in vitro fertilized using fresh epididymal sperm obtained from abattoir-derived
porcine testis and cultured in NSCU23 medium supplemented with 0.4% BSA.
The efficiency of transgenesis was monitored by visualization of green florescence
under UV illumination using an EGFP filter set. Results showed that the cleavage
rate of injected oocytes (68.7 Æ 0.5%) was similar to that of uninjected control
oocytes (67.8 Æ 0.4%) although a high percentage of injected oocytes showed
developmental block at the 2–4 cell stage. The EGFP expression rate at the 2–4
cell stage, when expressed as proportion of injected oocyte, was 17.2 Æ 0.1%.
Interestingly, mosaicism was not observed. The EGFP expression rate increased to
26.7 Æ 0.1% by increasing the DNA concentration to 40 ng/mL. Injecting the DNA
Nanoparticles for Gene Delivery into Stem Cells and Embryos
71
nanoSMGT, the ratios of linear-to-circular plasmid did not influence the uptake
by sperm cells and none of the tested treatments affected sperm motility and
viability after nanotransfection [130, 131]. Campos et al. [131] also used halloysite
clay nanotubes (HCN) for nanoSMGT. They observed that the mean number of
plasmids taken up by cattle sperm was higher in HCN-based gene delivery than by
using lipofection. IVF of oocytes with HCN-transfected sperm successfully resulted
in transgenic embryos with higher efficiency but, unfortunately, the transgene did
not express.
5.2 NP-Based Gene Delivery into Oocytes
Transgenic animal production via introduction of genes into oocytes have not been
very fruitful by conventional methods due to the high cytoplasmic content of
nuclease enzymes, cytoplasmic sequestration of injected DNA by DNA binding
proteins and cytoskeletal elements and the lack of DNA to transport across the
nuclear membrane. Some studies have shown that simultaneous introduction of
DNA along with the sperm can lead to successful production of transgenic mice
[132]. However, this method, called MII transgenesis, required intracytoplasmic
sperm injection to be performed and was not very fruitful in non-rodent species.
Use of lentriviral vectors, on the other hand, has resulted in successful production of
transgenic animals via gene introduction in oocytes.
MII-stage oocytes lack a nuclear membrane and offer a unique opportunity for
DNA to interact with the host chromatin to produce transgenic animals. However,
all efforts to produce transgenic animals via direct injection of DNA into oocytes
have failed. We have recently developed a NP-based gene delivery method for
mammalian oocytes, called oocyte-mediated gene transfer (OMGT) (Fig. 4). Pig
oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured
in vitro for 42–44 h and microinjected with DNA NP solution (10 ng/mL) using a
femtojet microinjector (Eppendorf, Hamburg, Germany). The DNA (4.7 kb) was
derived from the pEGFP-C1 plasmid (Clontech Laboratories, CA, USA), which
contains EGFP-encoding transgene under the control of cytomegalovirus (CMV)
promoter, and linearized with ApaLI restriction enzyme. Injected oocytes were then
in vitro fertilized using fresh epididymal sperm obtained from abattoir-derived
porcine testis and cultured in NSCU23 medium supplemented with 0.4% BSA.
The efficiency of transgenesis was monitored by visualization of green florescence
under UV illumination using an EGFP filter set. Results showed that the cleavage
rate of injected oocytes (68.7 Æ 0.5%) was similar to that of uninjected control
oocytes (67.8 Æ 0.4%) although a high percentage of injected oocytes showed
developmental block at the 2–4 cell stage. The EGFP expression rate at the 2–4
cell stage, when expressed as proportion of injected oocyte, was 17.2 Æ 0.1%.
Interestingly, mosaicism was not observed. The EGFP expression rate increased to
26.7 Æ 0.1% by increasing the DNA concentration to 40 ng/mL. Injecting the DNA
Nanoparticles for Gene Delivery into Stem Cells and Embryos
71