The ability to create transgenic livestock is a tremendous benefit in scientific
research for many disciplines including functional genomics, pharmaceutical synthesis
and development of enhanced production animals. Transgenes can either be stably or
transiently expressed to alter gene function and obtain a specifically engineered
phenotype. To create a transgenic bovine embryo, genetically altered somatic cells must
be used in somatic cell nucleus transfer, or early 1-cell embryos (zygotes) must be
microinjected with plasmid DNA or small interfering RNA (siRNA). Given the cost and
skill associated with both methods, a preliminary investigation exploring alternative
delivery techniques of siRNA (transient expression) into bovine zygotes with a nonhomologous
Cy3 labeled siRNA (Cy3-siRNA) was first performed. It was discovered
that zygotes injected with more than 50 Bmol L-1 of Cy3-siRNA fail to form a blastocoel
and that, although bovine zygotes are not susceptible to chemical transfection, the
trophectoderm cells of the blastocyst are. Based on this information, bovine E-cadherin
gene expression was compared in day 9 blastocysts derived from either injected zygotes (day 1) or transfected blastocysts (day 7) with a Cy3 labeled E-cadherin specific siRNA
(Cy3-siEcad) to determine 1) if gene suppression in zygotes injected with 25 Bmol L-1
Cy3-siEcad continues during embryo development up to hatching, and 2) if blastocysts
transfected at a ratio of 9:6 with GeneJammer® truly experience gene knock down after
siRNA transfection capable of maintaining suppression to day 9. Quantitative PCR
indicated blastocysts transfected with Cy3-siEcad had a significant 15.3% decrease (P <
0.05) in E-cadherin mRNA at day 9 compared to the injected zygotes. Protein
fluorescence analysis from immunocytochemistry of whole mounted day 9 blastocysts
revealed injected zygotes accumulated significantly less E-cadherin protein (67.7%) than
the transfected blastocysts (P < 0.05). From these data, it can be concluded that although
siRNA injection may be capable of knocking down gene expression for the first 7 days
of embryonic development, it does not persist to the hatching stage; however, blastocysts
transfected at day 7 do express altered gene expression in the trophectoderm which can
continue through embryonic hatching events.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-3048 |
| Date | 15 May 2009 |
| Creators | Hanna, Carol Bailey McCormick |
| Contributors | Kraemer, Duane C. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | electronic, application/pdf, born digital |
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