Title page for ETD etd-12172008-063219
|Type of Document
||Hajdu, Melissa Anne
||Effect of culture conditions, donor source, and injection site on in vitro development of deoxyribonucleic acid microinjected porcine zygotes
||Master of Science
|Knight, James W.
|Gwazdauskas, Francis C.
|Pearson, Ronald E.
|Velander, William H.
|Date of Defense
A series of experiments were used to evaluate three culture media and two
incubation temperatures for their ability to support development of DNA microinjected
porcine zygotes. Development in vitro was compared between embryos collected from
postpubertal and prepubertal donors and between embryos injected with DNA into the
pronucleus and the cytoplasm. Additionally, embryos were analyzed by the polymerase
chain reaction (PCR) for the presence of the transgene. One-cell embryos (n=458) were
recovered from 36 postpubertal gilts in Experiment 1. Injected and control embryos were
cultured in modified media NCSU-23 (mNCSU-23), NCSU-37 (mNCSU-37), and CZB at
37°C and 38.8°C for 7 d. In Experiment 2, one-cell embryos (n=245) were collected from
postpubertal (n=15) and prepubertal (n=14) gilts, microinjected with DNA, and cultured in
medium mNCSU-23. Superovulated prepubertal gilts (n=22) were flushed in Experiment
3 to yield 343 one-cell embryos which had DNA injected into the cytoplasm or
pronucleus. Whole embryos were assessed by PCR. Mean percentages of embryos
developing to the expanded or hatched blastocyst stage in mNCSU-23 and mNCSU-37
did not differ from each other (p>.05), but both were greater than the development in
CZB (p<.05). Development was greater at 38.8°C (p<.05) than at 37° C. Microinjection
of DNA decreased the developmental percentage (p<.05) from that of non-injected
controls. Embryos collected from postpubertal gilts had a higher percentage (68.0 ± 3.4)
of expanded and hatched blastocysts than embryos from prepubertal donors (29.0 ± 4.6,
p<.05). No difference was seen in development between embryos injected in the
pronucleus or cytoplasm (p>. 05), but development for both was less than for control
embryos (p<.05). Results of PCR analysis indicated that 40% of the embryos developing
to the expanded blastocyst stage were positive for the transgene compared to a rate of
60% positive for degenerate embryos. These studies show that DNA microinjected
porcine zygotes can be cultured to the expanded blastocyst stage in media mNCSU-23 and
mNCSU-37 at 38.8°C. Microinjection of DNA decreases survival of embryos collected
from both postpubertal and prepubertal sources, but postpubertal embryos exhibit a higher
rate of development. Cytoplasmic injection does not improve embryo viability in vitro
above that of pronuclear injection. Finally, whole embryo analysis by PCR is possible, but
cross specificity of human Protein C and whey acidic protein (WAP) oligonucleotides for
endogenous porcine DNA is strong and creates difficulty in applying PCR analysis to
embryos microinjected with WAP-PC transgenes.
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