Fast Quantitative Real-Time PCR-Based Screening for Common Chromosomal Aneuploidies in Mouse Embryonic Stem Cells

Chromosomal integrity has been known for many years to affect the ability of mouse embryonic stem cells (mESCs) to contribute to the germline of chimeric mice. Abnormal chromosomes are generally detected by standard cytogenetic karyotyping. However, this method is expensive, time consuming, and often omitted prior to blastocyst injection, consequently reducing the frequency of mESC-derived offspring. Here, we show a fast, accurate, and inexpensive screen for identifying the two most common aneuploidies (Trisomy 8 and loss of chromosome Y) in genetically manipulated mESCs using quantitative real-time PCR (qPCR). Screening against these two aneuploidies significantly increases the fraction of normal mESC clones. Our method is extremely sensitive and can detect as low as 10% aneuploidy among a large population of mESCs. It greatly expedites the generation of mutant mice and provides a quick tool for assessing the aneuploidy percentages of any mESC line

β2 Adrenergic Receptor Fluorescent Protein Fusions Traffic to the Plasma Membrane and Retain Functionality

Green fluorescent protein (GFP) has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs), Cerulean (and mCerulean3), Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the β2 adrenergic receptor (β2AR). We have characterized these β2AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane bound β2AR::XFP fusions robustly localized in the filopodia identical to gap::XFP fusions. All β2ARR::XFP fusions show responses indistinguishable from each other and the non-fused form after isoprenaline exposure. Our results provide a platform by which G-protein coupled receptors can be dissected for their functionality

β₂ Adrenergic Receptor Fluorescent Protein Fusions Traffic to the Plasma Membrane and Retain Functionality

<div><p>Green fluorescent protein (GFP) has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs), Cerulean (and mCerulean3), Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the β₂ adrenergic receptor (β₂AR). We have characterized these β₂AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane bound β₂AR::XFP fusions robustly localized in the filopodia identical to gap::XFP fusions. All β₂AR::XFP fusions show responses indistinguishable from each other and the non-fused form after isoprenaline exposure. Our results provide a platform by which G-protein coupled receptors can be dissected for their functionality.</p> </div

The transcriptional repressor Sum1p counteracts Sir2p in regulation of the actin cytoskeleton, mitochondrial quality control and replicative lifespan in Saccharomyces cerevisiae

Increasing the stability or dynamics of the actin cytoskeleton can extend lifespan in C. elegans and S. cerevisiae. Actin cables of budding yeast, bundles of actin filaments that mediate cargo transport, affect lifespan control through effects on mitochondrial quality control. Sir2p, the founding member of the Sirtuin family of lifespan regulators, also affects actin cable dynamics, assembly, and function in mitochondrial quality control. Here, we obtained evidence for novel interactions between Sir2p and Sum1p, a transcriptional repressor that was originally identified through mutations that genetically suppress sir2∆ phenotypes unrelated to lifespan. We find that deletion of SUM1 in wild-type cells results in increased mitochondrial function and actin cable abundance. Furthermore, deletion of SUM1 suppresses defects in actin cables and mitochondria of sir2∆ yeast, and extends the replicative lifespan and cellular health span of sir2∆ cells. Thus, Sum1p suppresses Sir2p function in control of specific aging determinants and lifespan in budding yeast

Visualization of OP 6 cells transiently expressing individual β₂AR::XFPs and mixed.

<p>Mixed OP 6 cells expressing [β₂AR::Cerulean, β₂AR::Venus, β₂AR::mCherry] in (A), [β₂AR::Teal, β₂AR::Venus, β₂AR::mCherry] in (B) and [β₂AR::GFP, β₂AR::mCherry, β₂AR::AFP] in (C). Each β₂AR::XFP fusion was solely identified in its respective channel (Ai, Cerulean, Bi, Venus, Ci, mCherry), (Aii, Teal, Bii, Venus, Cii, mCherry), (Aiii, GFP, Biii, mCherry, Ciii, AFP).</p

Colocalization of β₂AR::XFP and transferrin after isoprenaline exposure.

<p>Single OP 6 cells expressing each β₂AR::XFP construct were incubated with 20µg/ml of Alexa Fluor transferrin 647 (A-E) or Alexa Fluor transferrin 488 (F, G) for 30 minutes and then exposed to 10µM isoprenaline for 20 minutes. β₂AR::XFP is diffusely expressed at the plasma membrane, and transferrin is localized in the endosomes (A-G, 0) before isoprenaline exposure. After 20 minutes, β₂AR::XFP is internalized and becomes punctate, colocalizing with transferrin (arrowheads). ICQ values for all fusions reflect a significant increase (P<0.0001) in dependency after exposure to isoprenaline. ICQ values for 0 minute and 20 minute isoprenaline stimulation: (A) 0.086, 0.186 (B) 0.046, 0.187 (C) 0.116, 0.227 (D) -0.006, 0.212 (E) 0.132, 0.319 (F) 0.013, 0.254 (G) 0.047, 0.133.</p

Dose response curves for β₂AR::XFPs.

<p>Cells expressing each β₂AR::XFP and human Gα15 were exposed to concentrations of isoprenaline and analyzed using the FLIPR assay. Normalized curves (A) show an EC50 between 2.6-5.2 x 10^-9 for all fusions. Unnormalized curves (B) show a difference in maximum RFUs for individual fluorophore fusions. Transient expression of β₂AR::GFP truncated reveals no membrane expression (C) whereas β₂AR::GFP human localizes to filopodia (D, arrowhead).</p

Detailed time course analysis of β₂AR::Teal and β₂AR::Venus.

<p>ICQ values were used to measure colocalization events. (A) Single OP 6 cells (n=2) expressing the β₂AR::Teal or β₂AR::Venus construct were incubated with 20µg/ml of Alexa Fluor transferrin 647 for 30 minutes and then exposed to 10µM isoprenaline and imaged live every 4 minutes until 20 minutes. (B) Single OP 6 cells (n=3,4) expressing the β₂AR::Teal or β₂AR::Venus construct and myc-tagged β-Arrestin2 were fixed after 24 hours and incubated with rabbit polyclonal antibody for Myc after exposure to 10µM isoprenaline in 4 minute intervals between 0 and 20 minutes and were visualized with Anti-rabbit Alexa Fluor 546 antibody. Fusions of Teal and Venus to the β₂AR follow the same time course for internalization into the early endosomes (A) and the same time course regulation by β-Arrestin2 (B). Error bars represent standard error.</p

OP 6 cells transiently expressing gap::XFPs and β₂AR::XFPs.

<p>Single cells expressing each gap tagged fluorescent protein localizes to the plasma membrane (A-G). gap::XFPs also target to the filopodia (arrowheads, Ai-Gi magnified images). Single cells expressing each β₂AR fusion (β₂AR::XFP) (H-N) exhibit diffuse protein expression at the plasma membrane defined by localization at the filopodia (arrowheads, Hi-Ni magnified images). The ability of β₂AR to traffic to the plasma membrane is unaffected by fusion to the seven tested fluorescent proteins.</p

Filopodia count in OP 6 cells transiently expressing untagged fluorescent proteins, gap::XFPs, or β₂AR::XFPs.

<p>Filopodia on N=10 cells were counted for cells expressing each untagged fluorescent protein, tagged with gap, or fused to the β₂AR. gap::XFPs featured 78-141 filopodia per 10 cells, and β₂AR::XFPs featured 97-167 filopodia per 10 cells. Cells expressing the untagged fluorescent proteins featured between 7-16 filopodia. Expression in filopodia of gap::XFPs and β₂AR::XFPs was significantly different from the untagged XFPs, Fisher’s exact test P<0.0001.</p