Embryos derived by somatic cell nuclear transfer (SCNT) produce few
pregnancies that result in a live, healthy offspring. This has largely been attributed to the
aberrant reprogramming of the somatic cell DNA used for cloning. In order to improve
the efficiency of cloning there is a great deal of research needed to determine the role of
proteins involved in early embryonic reprogramming. In addition, studies are needed to
determine effects on somatic and embryonic cell development as a result of altering
these proteins.
In this study we investigate the use of RNA interference in bovine somatic cells
and embryos to knock down the expression of DNA methyltransferase-1 (DNMT1), an
enzyme responsible for maintenance methylation in mammalian cells. We designed our
experiments to test whether or not knocking down the DNMT1 gene would lead to a
decrease in global methylation. It is our hypothesis that using somatic cells with reduced
methylation may be advantageous for increasing the efficiency of cloning via somatic
cell nuclear transfer. To accomplish this task, we have designed an infectious non-replicating lentiviral
vector capable of delivering a gene that produces a short hairpin RNA targeting the
mRNA of DNMT1. The construct included a sequence coding for green fluorescent
protein (GFP) that will allow us to identify cells expressing the hairpin as well as a
region coding for neomycin resistance so we could select for a pure population of
transgenic cells to use for analysis.
Infecting bovine fetal fibroblast cells with genes encoding shRNAs that target
DNMT1 was successful. Quantitative real time PCR analysis of DNMT1 mRNA
suggests that our shRNAs are capable of an 80% knockdown. The protein blot of
indicates up to 90% knockdown of DNMT1. Cells transduced twice with a high titer
virus showed the highest knockdown of both DNMT1 mRNA and the protein. Analysis
of immunolabeled cytosine methylaiton showed a global decrease in DNA methylation
as a result of the DNMT1 knockdown. However, double transduced cells with a high
knockdown percentage of DNMT1 mRNA and protein became hypermethylated.
The second experiment was conducted to determine the effect of injecting small
interfering RNAs (siRNAs) targeting DNMT1 into oocytes prior to parthenogenic
activation. This experiment was designed to give us information on the survivability and
epigenetic profile of early embryos with decreased DNMT1. Oocytes injected with
siRNA targeting DNMT1 had little development past the 8-cell stage as compared to the
sham injected oocytes. This treatment group also had decreased DNA methylation as
determined by immunolabeling of methylated cytosine residues.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2009-05-623 |
| Date | 20 January 2010 |
| Creators | Stroud, Todd |
| Contributors | Long, Charles R. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Thesis |
| Format | application/pdf |
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