N Brossier is supported by the Francis S. Collins Scholars Program in Neurofibromatosis Clinical and Translational Research funded by the Neurofibromatosis Therapeutic Acceleration Program (NTAP, Grant # 210112). This publication was supported in part by funding from the NTAP at the Johns Hopkins University School of Medicine. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of The Johns Hopkins University School of Medicine. Additional support by the St. Louis Children&#39;s Hospital (FDN-2022-1082 to NMB) and the Washington University in St. Louis Diabetes Research Core (NIH P30 DK020579). Microscopy was performed through the use of the Washington University Center for Cellular Imaging (WUCCI), supported by the Washington University School of Medicine, The Children&#39;s Discovery Institute of Washington University, and St. Louis Children&#39;s Hospital (CDI-CORE-2015-505 and CDI-CORE-2019-813) and the Foundation for Barnes-Jewish Hospital (3770 and 4642). Nestin-CFPnuc35 mice were generously provided by Grigori Enikolopov (Renaissance School of Medicine, Stony Brook University, NY), and Nf1 mice heterozygous for either an R681X or C383X germline mutation32,38,39 were generously provided by David Gutmann (Washington University School of Medicine, St. Louis, MO). Figure 1 was created with BioRender.com.

All animal procedures in this study followed NIH guidelines and were approved by the Institutional Animal Care and Use Committee of Washington University in St. Louis. Animals were housed with standard 12 h light:dark cycling and free access to food and water.
1. Maternal diet
<ol>
	<li>Place female mice on control chow (CD) or obesogenic diet (Ob) at 4 weeks of age.</li>
	<li>At 8-12 weeks of age, set up timed mating by monitoring the mucus plug of the dam in the early morning to determine the correct collection age. To verify diet-induced obesity, weigh females at mating. 
	NOTE: A range of 8-12 weeks was used for the initial mating setup to ensure that the obesogenic diet-fed females were overweight compared to their counterparts, which did not always occur by 8 weeks of age. The day of the plug is considered embryonic day 1 (E1), and all the animals used in this study were collected at E19. The late fetal time point was chosen due to neurodevelopmental changes previously observed at this time in obesogenic diet-exposed animals (data not shown).</li>
</ol>
2. Fetus&#160;removal from the dam
<ol>
	<li>First, ensure that the dam appears gravid (abdominal shape and fullness, abdominal palpation, or fetal movement). If in doubt, weigh the female again to measure weight gain. 
	NOTE: Late-term pregnant mice should display at least 3-4 g of weight gain. To increase the likelihood of conception in the desired timeframe, dirty (urine- and pheromone-containing) sawdust from a male&#39;s cage can be added to the female&#39;s cage daily for 3 days prior to mating to induce estrus34.</li>
	<li>Euthanize the dam by placing it into a chamber with a paper towel containing 3 mL of isoflurane, being careful to avoid direct contact between dam and the anesthetic-soaked paper towel. After the animal is sedated, place it on a paper towel, hold it by the tail, place dissection scissors at the neck, and then force the scissors upward towards the head to perform cervical dislocation. Alternatively, perform decapitation after isoflurane sedation. 
	NOTE: Serum can be collected from the dam at this step, using a similar methodology as described below but with larger capillary tubes. If this is desired, decapitation rather than cervical dislocation should be performed. Care should be taken to ensure that the phenotype of interest is not altered by the administration of isoflurane immediately before euthanasia.</li>
	<li>Place the dam ventral side up in a 100 mm dish. Pinch the skin covering the abdomen and make a midline incision with dissection scissors. Make additional incisions perpendicular to the midline incision to create skin flaps to expose the uterus containing the embryos.</li>
	<li>Pull the uterus out of the abdominal cavity. Using scissors, disconnect the uterus from the dam. Place the intact embryonic sac (&#34;pearls on a string&#34;) containing the embryos into a dish with cold sterile 1x phosphate-buffered saline (PBS). Place the dish of fetuses on ice. 
	NOTE: As each litter typically contains 6-10 pups that will be harvested for both brains and serum, fetuses are kept on ice to avoid tissue degradation. Placing pups on ice additionally ensures adequate euthanasia of pups, as maternal euthanasia alone may not always be sufficient. If only serum collection is desired, room temperature collection may be preferred.</li>
</ol>
3. Fetal serum collection
<ol>
	<li>Remove the fetus from the embryonic sac by carefully dissecting with #5 forceps. Transfer the embryo to a dish using curved forceps with fresh cold sterile 1x PBS.</li>
	<li>Gently blot the animal on a paper towel to remove residual amniotic fluid and PBS from the specimen.</li>
	<li>Hold the animal by the abdomen. Partially decapitate the animal with micro scissors by making an incision at the ventral neck. Make sure the blood vessels are cut, but the skin keeping the head is still attached. 
	NOTE: The head must remain attached so that blood can be collected from the body and head simultaneously.</li>
	<li>Hold the body at a 45&#176; angle with the incision facing down. Hold the 50 &#181;L minivette (capillary tube) parallel to the incision site of the dam where the blood forms a droplet. Carefully allow the 50 &#181;L capillary tube to fill with blood. 
	NOTE: Capillary tube x2 or 100 &#181;L capillary tube can be used to collect up to ~75 &#181;L of blood.</li>
	<li>Place the head in a fresh 60 mm dish containing cold, sterile 1x PBS for further tissue collection (section 4).</li>
	<li>After the blood is within the capillary tube, press the plunger quickly into a 1.5 mL tube. Flick the tube or quickly spin it to make sure the blood is in the bottom of the tube.</li>
	<li>Repeat for the remainder of the litter. 
	NOTE: Keeping the pups on ice and moving quickly between is essential to reduce the risk of postmortem coagulation that will limit collection volume.</li>
	<li>Coagulate the blood at room temperature for 30 min.</li>
	<li>Centrifuge the blood samples at 4 &#176;C, 16,000 &#215; g&#160;for 20 min.</li>
	<li>Transfer the serum to fresh 500 &#181;L tubes with a 200 &#181;L pipette, without disturbing the blood cell pellet at the bottom of the tube. 
	NOTE: Serum should be straw-colored with no red debris. Each fetus should yield 15-30 &#181;L of serum.</li>
	<li>Store the collected serum at -80 &#176;C.</li>
</ol>
4. Fetal&#160;brain collection
<ol>
	<li>Looking through the dissecting microscope, hold the snout using curved forceps and use #5 forceps to carefully peel off the skin from the skull cap, keeping it attached at the snout for ease of holding. Angle #5 forceps to 45&#176; relative to the cranium, carefully puncture the clear membrane that is the developing skull, shift the forceps parallel to the cranium, insert it underneath the skull cap, and then pinch and peel to one side. After one side is removed, grasp the remaining side with #5 forceps and peel to the other side.</li>
	<li>Gently scoop the brain out of the skull using a micro spoon and transfer the brain to a fresh 60 mm dish containing cold 1x PBS. 
	NOTE: At this point, whole brains can be fixed and embedded for immunostaining. See previously published methodology32.</li>
	<li>If tissue must be snap-frozen for other molecular applications, sub-dissect the area of interest. Isolation of the periventricular tissue around the lateral, third, and fourth ventricles is described below.
	<ol>
		<li>Using the curved forceps, hold the brain at the junction of the hindbrain and forebrain with the non-dominant hand. With the dominant hand, hold the scalpel with the blade parallel to the surface of the brain. Press down firmly at the mid-cortical region (Figure 1).</li>
		<li>Still holding the hindbrain, make a second incision in front of the hindbrain to separate it from the forebrain (Figure 1). In the anterior-most section, look for two small semicircular spaces located within the horizontal plane, which are the lateral ventricles. Use the curved forceps placed in the center of each ventricle to hold the brain in place, carefully dissect the lining using #5 forceps, and remove to a fresh clean 1.5 mL microcentrifuge tube.</li>
		<li>In the middle section, look for a small linear space on the ventral surface, which is the third ventricle. Using #5 forceps, carefully pinch off the lining on either side of the ventricular surface, then remove to a fresh clean 1.5 mL microcentrifuge tube.</li>
		<li>In the posterior-most section, look for a small linear space central within the hindbrain, which is the fourth ventricle. Dissect the lining on both sides using #5 forceps, then remove to a fresh clean 1.5 mL microcentrifuge tube. 
		NOTE: If slicing the hindbrain is not a clean cut, the fourth ventricle tissue may be obscured and/or destroyed.</li>
		<li>For steps 4.3.2-4.3.4, immediately after the tissue has been placed into microcentrifuge tubes, place the tubes quickly into a cryo-safe container containing liquid nitrogen. 
		NOTE: Tubes can be kept in liquid nitrogen until the remaining samples have been dissected. CAUTION: Liquid nitrogen can cause severe frostbite. Do not stick hands directly in liquid nitrogen and avoid splashes.</li>
		<li>Place frozen tube(s) on dry ice and transfer frozen tissue into a -80 &#176;C freezer until use.</li>
	</ol>
	</li>
</ol>

Simultaneously Harvesting the Brain and Serum from the Murine Fetus

The authors do not have any conflicts of interest.

Traditional methods for collecting blood from mice include retrobulbar, tail vein, saphenous vein, facial vein, and jugular vein bleeding40,41,42. Unfortunately, these methods are not ideal for embryonic blood collection due to the size of the animal and small, delicate vasculature. Collection of blood via gravity-aided drainage after decapitation led to both low collection volumes and significant hemolysis in our samples. Previ...

Neurofibromatosis Type 1 (NF1) is considered a RASopathy, a group of disorders characterized by germline genetic mutations resulting in activation of the RAS/MAPK (RAt Sarcoma virus/Mitogen-activated Protein Kinase) signaling pathway. Patients with the NF1 RASopathy are at risk for developing many different manifestations, including both benign and malignant tumors of the central (optic pathway glioma1,2, high-grade glioma3,4) and peripheral (plexiform neurofibroma5,6, malignant peripheral nerve sheath tumor7,8) nervous system as well as bony dysplasias9 and skin pigmentary abnormalities10 (axillary freckling, caf&#233;-au-lait macules). The effect of this disorder on cognition and neurodevelopment is increasingly being recognized, with NF1 patients displaying an increased incidence of learning deficits, hyperactivity, and autism spectrum disorder11,12,13. However, there is significant heterogeneity in the development of these phenotypes between patients13,14,15,16,17, and it is unclear why some patients display significant cognitive impairments while others are unaffected. Maternal diet-induced obesity has been shown to similarly affect learning and behavior in the general population18,19,20,21,22,23,24,25,26,27,28, suggesting that differential maternal dietary exposures in NF1 could be one source of this clinical heterogeneity. In particular, children of obese mothers display an increased risk of developing hyperactivity18,19,20,23,25,26, autism19,24,27, executive function deficits21,23, and have lower full-scale IQ scores22,28. However, patients with NF1 have altered metabolic phenotypes compared to the general population, including decreased incidence of obesity and diabetes29,30,31, making it unclear whether they would respond similarly to dietary stimuli.
To address these questions, we wished to determine whether obesogenic-diet-induced changes to the macronutrient profile in fetal offspring with Nf1 contributed to neurodevelopmental changes. We have previously collected high-quality whole and regionally micro-dissected tissue appropriate for neurodevelopmental applications from the fetal brain32. However, fetal blood collection is challenging due to the small body size and low blood volume33. The collection of blood via gravity-aided drainage after decapitation led to low collection volumes and significant hemolysis in our samples, which can affect downstream application interpretation. Collection via aspiration from the fetal heart or thoracic vessels, as has been previously reported33, was technically challenging and also resulted in frequent hemolysis. We thus developed a method for fetal serum collection, which utilizes specialized capillary tubes to allow for higher volume collection without significant shear stress.
Here, we present this method to simultaneously collect embryonic brains and fetal serum from Nf1-heterozygous pups exposed to a high-fat, high-sugar diet versus a control diet in utero (Figure&#160;1 and Supplemental Table S1). Brains were cryo-embedded for subsequent analysis by immunofluorescence or regionally micro-dissected and flash-frozen for subsequent use in molecular biology applications. High-quality serum was obtained, suitable for downstream applications such as macronutrient profiling. Utilizing this method, we identified that maternal high-fat, high-sucrose dietary exposure leads to the elevation of serum cholesterol levels in both WT and Nf1-heterozygous pups.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#5/45 Forceps</td>
			<td>Dumont&#160;</td>
			<td>11251-35</td>
			<td>tip shape: angled 45&#176;</td>
		</tr>
		<tr>
			<td>4200 Tapestation</td>
			<td>Agilent</td>
			<td>G2991BA</td>
			<td>Verify RNA integrity and quality, measurement of RIN values</td>
		</tr>
		<tr>
			<td>Benchtop Liquid Nitrogen Container</td>
			<td>Thermo Fisher</td>
			<td>2122</td>
			<td>Or other cryo-safe container</td>
		</tr>
		<tr>
			<td>Control Chow</td>
			<td>PicoLab</td>
			<td>5053</td>
			<td>Research diets D12328 (low-fat, low-sugar) may also be used.</td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>Cole Parmer</td>
			<td>UX-10818-25</td>
			<td>Tip shape: curved 90&#176;</td>
		</tr>
		<tr>
			<td>Dissecting blade handle</td>
			<td>Cole-Parmer Essentials</td>
			<td>10822-20</td>
			<td>SS Siegel-Type, #10 to #15 blades</td>
		</tr>
		<tr>
			<td>EMS SuperCut Dissection Scissors</td>
			<td>Electron microscope sciences</td>
			<td>72996-01</td>
			<td>5&#189;&#34; (139.7 mm), Straight</td>
		</tr>
		<tr>
			<td>GFAP Antibody</td>
			<td>Abcam</td>
			<td>ab7260</td>
			<td>Dilute 1:350. Block with 10% serum containing 0.3 M Glycine.</td>
		</tr>
		<tr>
			<td>Glassvan Carbon Steel Surgical Blades, Size 11</td>
			<td>MYCO medical</td>
			<td>2001T-11</td>
			<td>#11 blades allow straight, flat cut</td>
		</tr>
		<tr>
			<td>Micro lab spoon</td>
			<td>Az Scilab</td>
			<td>A2Z-VL001</td>
			<td>stainless steel, autoclavable</td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>Rubis</td>
			<td>78180-1C3</td>
			<td>model 1C300</td>
		</tr>
		<tr>
			<td>Minivette&#160;POCT neutral</td>
			<td>Sarstedt</td>
			<td>17.2111.050</td>
			<td>nominal volume: 50 &#181;L, without preparation</td>
		</tr>
		<tr>
			<td>Nanorop</td>
			<td>Thermo Fisher</td>
			<td>13-400-519</td>
			<td>Measure RNA concentration, 260/280 ratios</td>
		</tr>
		<tr>
			<td>Obesogenic diet</td>
			<td>Researchdiets.com</td>
			<td>D12331</td>
			<td>High-fat, high-sucrose</td>
		</tr>
		<tr>
			<td>Total Cholesterol Reagent</td>
			<td>Thermo Fisher</td>
			<td>TR13421</td>
			<td>Colorimetric detection</td>
		</tr>
		<tr>
			<td>&#946;-actin antibody</td>
			<td>Cell Signaling</td>
			<td>8457</td>
			<td>Dilute 1:1,000.</td>
		</tr>
	</tbody>
</table>

concurrent collection of fetal murine brain and serum to assess effects of maternal diet on nutrition and neurodevelopment in neurofibromatosis type 1

Maternal diet-induced obesity has been demonstrated to alter neurodevelopment in offspring, which may lead to reduced cognitive capacity, hyperactivity, and impairments in social behavior. Patients with the clinically heterogeneous genetic disorder Neurofibromatosis Type 1 (NF1) may present with similar deficits, but it is currently unclear whether environmental factors such as maternal diet influence the development of these phenotypes, and if so, the mechanism by which such an effect would occur. To enable evaluation of how maternal obesogenic diet exposure affects systemic factors relevant to neurodevelopment in NF1, we have developed a method to simultaneously collect non-hemolyzed serum and whole or regionally micro-dissected brains from fetal offspring of murine dams fed a control diet versus a high-fat, high-sucrose diet. Brains were processed for cryosectioning or flash frozen to use for subsequent RNA or protein isolation; the quality of the collected tissue was verified by immunostaining. The quality of the serum was verified by analyzing macronutrient profiles. Using this technique, we have identified that maternal obesogenic diet increases fetal serum cholesterol similarly between WT and Nf1-heterozygous pups.

To illustrate the quality of brain tissue obtained via this technique, we show sample fetal brains from Nestin-CFPnuc mice35, immunostained for GFAP per a previously reported technique32. Nestin+ cells are seen lining the lateral ventricle (Figure 2A), with GFAP+ filaments extending from the surface. We did not observe differences between Nestin or GFAP expression in the lateral ventricle of CD versus Ob-exposed mice in ei...

Read this Scientific Article Protocol about Concurrent Collection of Fetal Murine Brain and Serum to Assess Effects of Maternal Diet on Nutrition and Neurodevelopment in Neurofibromatosis Type 1 at Jo

Author Spotlight: Collecting the Brain and Serum from the Same Mice Fetus to Study Brain Tumor Development

In this protocol, we describe a method for simultaneous collection of fetal brain tissue as well as high-quality, non-hemolyzed serum from the same mouse embryo. We have utilized this technique to interrogate how maternal dietary exposure affects macronutrient profiles and fetal neurodevelopment in mice heterozygous for Nf1 (Neurofibromatosis Type 1).

Concurrent Collection of Fetal Murine Brain and Serum to Assess Effects of Maternal Diet on Nutrition and Neurodevelopment in Neurofibromatosis Type 1

Maternal diet-induced obesity has been demonstrated to alter neurodevelopment in offspring, which may lead to reduced cognitive capacity, hyperactivity, ...

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430 Views.  Washington University in St. Louis. In this protocol, we describe a method for simultaneous collection of fetal brain tissue as well as high-quality, non-hemolyzed serum from the same mouse embryo. We have utilized this technique to interrogate how maternal dietary exposure affects macronutrient profiles and fetal neurodevelopment in mice heterozygous for Nf1 (Neurofibromatosis Type 1).

Video: Author Spotlight: Collecting the Brain and Serum from the Same Mice Fetus to Study Brain Tumor Development

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse splenocytes with the pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS), ODN1826, and heat-killed Listeria monocytogenes and examine their effect on the molecular circadian clock. Previously, studies have focused on examining the influence of LPS on the molecular clock using a variety of in vivo and ex vivo approaches from an assortment of models (e.g., mouse, rat, and human). This protocol describes the isolation and challenge of splenocytes, as well as the methodology to assess clock gene expression post-challenge via quantitative PCR. This approach can be used to assess not only the influence of microbial components on the molecular clock but other molecules as well that may alter expression of the clock. This approach could be utilized to tease apart the molecular mechanism of how PAMP-Toll-like receptor interaction influences clock expression.

This protocol describes a technique using mouse splenocytes to discover pathogen-associated molecular patterns that alter molecular clock gene expression.

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

Culturing and Manipulation of O9-1 Neural Crest Cells

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.

O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing.

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.