A multiplex PCR genotyping assay to distinguish XX and XY tissues in sheep.

Sex is an important biological variable as many physiological as well as disease processes differ between females and males. The fundamental biological distinction between females and males starts with chromosomal sex, and the establishment of XX and XY cells and tissues. Polymerase Chain Reaction (PCR) is a simple and effective method to easily determine chromosomal or genetic sex of cells and tissues. The goal of this study was to develop a simple multiplex PCR genotyping assay to distinguish XX and XY tissues in sheep. Primers were designed to amplify a fragment of the autosomal gene myogenin (MYOG) and sex determining region on the Y chromosome (SRY). PCR analysis was performed on a variety of genomic DNA samples isolated from fetal sheep skeletal muscle, brain, liver, and placenta, and revealed a single 259 bp band for MYOG in XX females, and a 259 bp band for MYOG and a 167 bp band for SRY in XY males. Amplicons were clearly distinguishable by gel electrophoresis, and their sequences revealed 100% identity to the known ovine MYOG and SRY sequence. The reported multiplex PCR genotyping assay provides a rapid means to distinguish XX and XY sheep tissues using low volume samples.

Knockout of the neonatal Fc receptor alters immune complex trafficking and lysosomal function in cultured podocytes.

Podocytes are key to preventing the filtration of serum proteins into the urine. Recent evidence also suggests that in immune mediated kidney diseases, podocytes are the targets of immune complexes (ICs). The mechanisms whereby podocytes handle and respond to ICs remain unknown. The neonatal Fc receptor (FcRn) is involved in IgG handling in podocytes and is also required in dendritic cells to traffic ICs to the lysosome for proteolytic degradation of antigen and presentation on MHC II. Here we examine the role of FcRn in handling ICs in podocytes. We show that knockout of FcRn in podocytes results in decreased trafficking of ICs to the lysosome and increases IC trafficking to recycling endosomes. FcRn KO also alters lysosomal distribution, decreases lysosomal surface area and decreases cathepsin B expression and activity. We demonstrate that signaling pathways in cultured podocytes differ after treatment with IgG alone versus ICs and that podocyte proliferation in both WT and KO podocytes is suppressed by IC treatment. Our findings suggest that podocytes respond differentially to IgG versus ICs and that FcRn modifies the lysosomal response to ICs. Elucidating the mechanisms underlying podocyte handling of ICs may provide novel pathways to modulate immune mediated kidney disease progression.

Placenta specific gene targeting to study histone lysine demethylase and androgen signaling in ruminant placenta.

Reproductive efficiency is critically dependent on embryo survival, establishment of a successful pregnancy and placental development. Recent advances in gene editing technology have enabled investigators to use gene knockdown and knockout approaches to better understand the role of hormone signaling in placental function and fetal growth and development. In this review, an overview of ruminant placentation will be provided, including recent data highlighting the role of histone lysine demethylase 1A and androgen signaling in ruminant placenta and pregnancy. Studies in ruminant placenta establish a role for histone lysine demethylase 1A in controlling genetic networks necessary for important cellular events such as cell proliferation and angiogenesis, as well as androgen receptor signaling during early placentation.