Melanocortins (MCs) play an important role in feeding, metabolism, and energy expenditure. While melanocortin receptor (MCR) mRNA has been found in the mesolimbic dopamine (DA) pathway, the ability of melanocortins to regulate feeding and other behaviors through actions on the mesolimbic DA system have not been examined. Short-hairpin RNAs (shRNAs) were created targeting MC3R and MC4R and were tested via in vitro studies for their ability to knockdown their target receptor. A total of three shRNAs were created targeting each receptor, and each shRNA caused successful knockdown. These shRNAs are tools that can be used for future in vivo studies to examine the various behavioral effects of melanocortins on the mesolimbic DA pathway.

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.

No description available.

Phox2a and Phox2b are two homeodomain proteins that control the differentiation of noradrenergic neurons during embryogenesis. In the present study, we examined the possible effect of Phox2a/2b on the in vitro expression of the norepinephrine transporter (NET) and dopamine β-hydroxylase (DBH), two important markers of the noradrenergic system. SK-N-BE(2)C cells were transfected with cDNAs or short hairpin RNAs specific to the human Phox2a and Phox2b genes. Transfection of 0.1 to 5 μg of cDNAs of Phox2a or Phox2b significantly increased mRNA and protein levels of NET and DBH in a concentration-dependent manner. As a consequence of the enhanced expression of NET after transfection, there was a parallel increase in the uptake of [ 3H]norepinephrine. Co-transfection of Phox2a and Phox2b did not further increase the expression of noradrenergic markers when compared with transfection of either Phox2a or Phox2b alone. Transfection of shRNAs specific to Phox2a or Phox2b genes significantly reduced mRNA and protein levels of NET and DBH after shutdown of endogenous Phox2, which was accompanied by a decreased [3H]norepinephrine uptake. Furthermore, there was an additive effect after cotransfection with both shRNAs specific to Phox2a or Phox2b genes on NET mRNA levels. Finally, the reduced DBH expression caused by the shRNA specific to Phox2a could be reversed by transfection with Phox2b cDNA and vice versa. The present findings verify the determinant role of Phox2a and Phox2b on the expression and function of NET and DBH in vitro. Further clarifying the regulatory role of these two transcription factors on key proteins of the noradrenergic system may open a new avenue for therapeutics of aging-caused dysfunction of the noradrenergic system.

No description available.