<meta charset="utf8"></head><body>採血とホルモン分析によるマーモセットの卵巣周期の評価

<ol>
	<li>Kholkute, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+progesterone+levels+throughout+the+ovarian+cycle+of+the+common+marmoset+(Callithrix+jacchus).">Plasma progesterone levels throughout the ovarian cycle of the common marmoset (Callithrix jacchus).</a> Primates. 25 (1), 123-126 (1984).</li><li>Harding, R. D., Hulme, M. J., Lunn, S. F., Henderson, C., Aitken, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+progesterone+levels+throughout+the+ovarian+cycle+of+the+common+marmoset+(Callithrix+jacchus).">Plasma progesterone levels throughout the ovarian cycle of the common marmoset (Callithrix jacchus).</a> J Med Primatol. 11 (1), 43-51 (1982).</li><li>Gilchrist, R. B., Wicherek, M., Heistermann, M., Nayudu, P. L., Hodges, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+follicle-stimulating+hormone+and+follicle+populations+during+the+ovarian+cycle+of+the+common+marmoset.">Changes in follicle-stimulating hormone and follicle populations during the ovarian cycle of the common marmoset.</a> Biol Reprod. 64 (1), 127-135 (2001).</li><li>Gromoll, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+subclass+of+the+luteinizing+hormone/chorionic+gonadotropin+receptor+lacking+exon+10+messenger+RNA+in+the+New+World+monkey+(Platyrrhini)+lineage.">A new subclass of the luteinizing hormone/chorionic gonadotropin receptor lacking exon 10 messenger RNA in the New World monkey (Platyrrhini) lineage.</a> Biol Reprod. 69 (1), 75-80 (2003).</li><li>M&#252;ller, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chorionic+gonadotrophin+beta+subunit+mRNA+but+not+luteinising+hormone+beta+subunit+mRNA+is+expressed+in+the+pituitary+of+the+common+marmoset+(Callithrix+jacchus).">Chorionic gonadotrophin beta subunit mRNA but not luteinising hormone beta subunit mRNA is expressed in the pituitary of the common marmoset (Callithrix jacchus).</a> J Mol Endocrinol. 32 (1), 115-128 (2004).</li><li>Pacchiarotti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+stimulation+protocol+in+IVF:+an+up-to-date+review+of+the+literature.">Ovarian stimulation protocol in IVF: an up-to-date review of the literature.</a> Curr Pharm Biotechnol. 17 (4), 303-315 (2016).</li><li>Ezcurra, D., Humaidan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+luteinising+hormone+and+human+chorionic+gonadotropin+when+used+in+assisted+reproductive+technology.">A review of luteinising hormone and human chorionic gonadotropin when used in assisted reproductive technology.</a> Reprod Biol Endocrinol. 12, 95 (2014).</li><li>Lopez-Gatius, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+response+to+prostaglandin+F(2alpha)+in+lactating+dairy+cows:+A+clinical+update.">Ovarian response to prostaglandin F(2alpha) in lactating dairy cows: A clinical update.</a> J Reprod Dev. 68 (2), 104-109 (2022).</li><li>Marini, R. P., Wachtman, L. M., Tardif, S. D., Mansfield, K., Fox, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Common Marmoset in Captivity and Biomedical Research. , (2018).</li><li>Schultz-Darken, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+collection+and+restraint+techniques+used+for+common+marmosets+(Callithrix+jacchus).">Sample collection and restraint techniques used for common marmosets (Callithrix jacchus).</a> Comp Med. 53 (4), 360-363 (2003).</li><li>Hopper, J., Kubik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+marmosets.">Common marmosets.</a> Handbook of Exotic Pet. , 27-42 (2020).</li><li>Harlow, C. R., Hearn, J. P., Hodges, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovulation+in+the+marmoset+monkey:+endocrinology,+prediction+and+detection.">Ovulation in the marmoset monkey: endocrinology, prediction and detection.</a> J Endocrinol. 103 (1), 17-24 (1984).</li><li>Ludlage, E., Mansfield, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+care+and+diseases+of+the+common+marmoset+(Callithrix+jacchus).">Clinical care and diseases of the common marmoset (Callithrix jacchus).</a> Comp Med. 53 (4), 369-382 (2003).</li><li>Summers, P. M., Wennink, C. J., Hodges, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloprostenol-induced+luteolysis+in+the+marmoset+monkey+(Callithrix+jacchus).">Cloprostenol-induced luteolysis in the marmoset monkey (Callithrix jacchus).</a> J Reprod Fertil. 73 (1), 133-138 (1985).</li><li>Takahashi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+of+healthy+offspring+following+ICSI+in+in+vitro-matured+common+marmoset+(Callithrix+jacchus)+oocytes.">Birth of healthy offspring following ICSI in in vitro-matured common marmoset (Callithrix jacchus) oocytes.</a> PLoS One. 9 (4), e95560 (2014).</li><li>Tomioka, I., Takahashi, T., Shimada, A., Yoshioka, K., Sasaki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+of+common+marmoset+(Callithrix+jacchus)+offspring+derived+from+in+vitro-matured+oocytes+in+chemically+defined+medium.">Birth of common marmoset (Callithrix jacchus) offspring derived from in vitro-matured oocytes in chemically defined medium.</a> Theriogenology. 78 (7), 1487-1493 (2012).</li><li>Abe, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+marmoset+genome+engineering+by+autologous+embryo+transfer+and+CRISPR/Cas9+technology.">Efficient marmoset genome engineering by autologous embryo transfer and CRISPR/Cas9 technology.</a> Sci Rep. 11 (1), 20234 (2021).</li><li>Summers, P. M., Shephard, A. M., Taylor, C. T., Hearn, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+cryopreservation+and+transfer+on+embryonic+development+in+the+common+marmoset+monkey,+Callithrix+jacchus.">The effects of cryopreservation and transfer on embryonic development in the common marmoset monkey, Callithrix jacchus.</a> J Reprod Fertil. 79 (1), 241-250 (1987).</li><li>Thomson, J. A., Kalishman, J., Hearn, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsurgical+uterine+stage+preimplantation+embryo+collection+from+the+common+marmoset.">Nonsurgical uterine stage preimplantation embryo collection from the common marmoset.</a> J Med Primatol. 23 (6), 333-336 (1994).</li><li>Hanazawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+invasive+transabdominal+collection+of+preimplantation+embryos+from+the+common+marmoset+monkey+(Callithrix+jacchus).">Minimally invasive transabdominal collection of preimplantation embryos from the common marmoset monkey (Callithrix jacchus).</a> Theriogenology. 78 (4), 811-816 (2012).</li><li>Ishibashi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+embryo+transfer+in+the+common+marmoset+monkey+(Callithrix+jacchus)+with+a+reduced+transfer+volume:+a+non-surgical+approach+with+cryopreserved+late-stage+embryos.">Efficient embryo transfer in the common marmoset monkey (Callithrix jacchus) with a reduced transfer volume: a non-surgical approach with cryopreserved late-stage embryos.</a> Biol Reprod. 88 (5), 115 (2013).</li><li>Kishimoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+novel+common+marmoset+embryonic+stem+cell+lines+under+various+conditions.">Establishment of novel common marmoset embryonic stem cell lines under various conditions.</a> Stem Cell Res. 53, 102252 (2021).</li><li>Diehl, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood,+including+routes+and+volumes.">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> J Appl Toxicol. 21 (1), 15-23 (2001).</li><li>Daskalaki, M., Drummer, C., Behr, R., Heistermann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+alfaxalone+for+short-term+anesthesia+can+confound+serum+progesterone+measurements+in+the+common+marmoset:+a+case+report.">The use of alfaxalone for short-term anesthesia can confound serum progesterone measurements in the common marmoset: a case report.</a> Primate Biol. 9 (2), 23-28 (2022).</li><li>Hodges, J. K., Cottingham, P. G., Summers, P. M., Liang, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+ovulation+in+the+marmoset+monkey+(Callithrix+jacchus)+with+human+chorionic+gonadotropin+following+prostaglandin-induced+luteal+regression.">Controlled ovulation in the marmoset monkey (Callithrix jacchus) with human chorionic gonadotropin following prostaglandin-induced luteal regression.</a> Fertil Steril. 48 (2), 299-305 (1987).</li><li>Barrett, J., Abbott, D. H., George, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extension+of+reproductive+suppression+by+pheromonal+cues+in+subordinate+female+marmoset+monkeys,+Callithrix+jacchus.">Extension of reproductive suppression by pheromonal cues in subordinate female marmoset monkeys, Callithrix jacchus.</a> J Reprod Fertil. 90 (2), 411-418 (1990).</li><li>Barrett, J., Abbott, D. H., George, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+cues+and+the+suppression+of+reproduction+in+subordinate+female+marmoset+monkeys,+Callithrix+jacchus.">Sensory cues and the suppression of reproduction in subordinate female marmoset monkeys, Callithrix jacchus.</a> J Reprod Fertil. 97 (1), 301-310 (1993).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AIA-360</td>
			<td>Tosoh Corporation</td>
			<td>0019945</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>AIA-PACK DILUENT CONCENTRATE</td>
			<td>Tosoh Corporation</td>
			<td>0020956</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>AIA-PACK SUBSTRATE SET II</td>
			<td>Tosoh Corporation</td>
			<td>0020968</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>AIA-PACK WASH CONCENTRATE</td>
			<td>Tosoh Corporation</td>
			<td>0020955</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>CMS-1</td>
			<td>CLEA Japan</td>
			<td></td>
			<td>Marmoset food</td>
		</tr>
		<tr>
			<td>Estrumate</td>
			<td>MSD Animal Health</td>
			<td></td>
			<td>PGF2alpha analog (cloprostenol)</td>
		</tr>
		<tr>
			<td>Gonal-f Subcutaneous Injection 150</td>
			<td>Merck Biopharma Co., Ltd.</td>
			<td></td>
			<td>FSH</td>
		</tr>
		<tr>
			<td>Gonatropin for intramuscular injection 1000</td>
			<td>ASKA Pharmaceutical Co., Ltd.</td>
			<td>872413</td>
			<td>hCG</td>
		</tr>
		<tr>
			<td>Heparin sodium injection solution 5,000 units/5 mL</td>
			<td>Mochida Pharmaceutical Co., Ltd.</td>
			<td>224122458</td>
			<td>Blood collection</td>
		</tr>
		<tr>
			<td>Immunochromatographic Test Kit for Detection of Common Marmoset Chorionic Gonadotropin (Dual Checker)</td>
			<td>CLEA Japan, Inc.</td>
			<td></td>
			<td>Determining CG level</td>
		</tr>
		<tr>
			<td>Low-profile double-arm microscope illumination LPF-SD</td>
			<td>SHIOKAZE GIKEN</td>
			<td></td>
			<td>Desk lamp for blood collection</td>
		</tr>
		<tr>
			<td>Marmoset blood collection restraint device</td>
			<td>JIC Japan</td>
			<td>JM-1006</td>
			<td>Blood collection 
			http://www.jic-japan.jp/prd/marmoset/prd016.html 
			email: vi@jic-japan.jp</td>
		</tr>
		<tr>
			<td>Metacam 0.05%</td>
			<td>Boehringer Ingelheim Animal Health Japan Co., Ltd.</td>
			<td></td>
			<td>Hematoma treatment</td>
		</tr>
		<tr>
			<td>Sample Cup, 3 mL, PS, for Tosoh 360 and AIA-600 II, 1000/Bag</td>
			<td>Globe Scientific</td>
			<td>110913</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>ST AIA-PACK iE2</td>
			<td>Tosoh Corporation</td>
			<td>0025224</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>ST AIA-PACK iE2 CALIBRATOR SET</td>
			<td>Tosoh Corporation</td>
			<td>0025324</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>ST AIA-PACK iE2 SAMPLE DILUTING SOLUTION</td>
			<td>Tosoh Corporation</td>
			<td>0025524</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>ST AIA-PACK PROGIII</td>
			<td>Tosoh Corporation</td>
			<td>0025240</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>ST AIA-PACK PROGIII CALIBRATOR SET</td>
			<td>Tosoh Corporation</td>
			<td>0025340</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>ST AIA-PACK PROGIII SAMPLE DILUTING SOLUTION</td>
			<td>Tosoh Corporation</td>
			<td>&#160;0025540</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>Syringe with 25 G (0.50 x 25 mm) needle</td>
			<td>TERUMO</td>
			<td>SS-01T2525</td>
			<td>Blood collection</td>
		</tr>
		<tr>
			<td>Tensolvet 5.000 I.E. gel.</td>
			<td>Dechra Pharmaceuticals</td>
			<td>14033492</td>
			<td>Hematoma treatment</td>
		</tr>
		<tr>
			<td>TOSOH MULTI-CONTROL SET</td>
			<td>Tosoh Corporation</td>
			<td>0015965</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
	</tbody>
</table>

blood sampling and hormone measurement for determining the stage in the ovarian cycle in marmosets

<meta charset="utf8"></head><body>著者スポットライト:生殖細胞研究と遺伝子組み換え技術による霊長類学の進歩

マーモセットの卵巣周期の病期を決定するための採血とホルモン測定

Common marmosets are small New World monkeys. Since many of their biological mechanisms are similar to those of humans, marmosets are potentially useful ...

blood-sampling-hormone-measurement-for-determining-stage-ovarian

Research

JoVE Journal

Biology

Blood Sampling and Hormone Measurement for Determining the Stage in the Ovarian Cycle in Marmosets

685 ビュー.  National Center for Child Health and Development. 私たちは、大きく分けて2つの側面に取り組んでいます。まず、ヒトを含む霊長類の雄性生殖細胞の理解を目指します。精原幹細胞とDNAメチル化の確立に着目しています。2つ目は、遺伝子組み換えマーモセットの新たな作製技術の開発です。遺伝子組み換えマーモセットの作製には、大変な労力が必要です。その課題の一つが、遺伝子組み?...

ビデオ: 著者スポットライト:生殖細胞研究と遺伝子組み換え技術による霊長類学の進歩

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

の精製 M. magneticum株AMB - 1 Magnetosome関連タンパク質MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

生物学

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.

モルモットにおける非ターミナル採血テクニック

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

ラットの外側尾静脈から血液をサンプリング

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

ギャップ結合細胞間コミュニケーション：毒物および毒素の有害効果を評価するための機能バイオマーカー、および天然物の健康上の利点

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive&#160;strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

小さいテレオストモデル日本メダカにおけるゴナデクトミーと血液サンプリング手順 (オリジアス ラティプ )

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

単核RNAシーケンシングのための成体マウス腎臓からの核分離

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Evidence for EpCAM and Cytokeratin Expressing Epithelial Cells in Normal Human and Murine Blood and Bone Marrow

Epithelial cells have been identified in the blood and bone marrow of patients with cancer and other diseases. However, the presence of normal epithelial cells in the blood and bone marrow of healthy individuals has yet to be identified in a consistent way. Presented here is a reproducible method for isolating epithelial cells from healthy human and murine blood and bone marrow (BM) using flow cytometry and immunofluorescence (IF) microscopy. Epithelial cells in healthy individuals were first identified and isolated via flow cytometry using epithelial cell adhesion molecule (EpCAM). These EpCAM+ cells were confirmed to express keratin using immunofluorescence microscopy in Krt1-14;mTmG transgenic mice. Human blood samples had 0.18% &#177; 0.0004 EpCAM+ cells (SEM; n=7 biological replicates, 4 experimental replicates). In human BM, 3.53% &#177; 0.006 (SEM; n=3 biological replicates, 4 experimental replicates) of mononuclear cells were EpCAM+. In mouse blood, EpCAM+ cells constituted 0.45% &#177; 0.0006 (SEM; n=2 biological replicates, 4 experimental replicates), and in mouse BM, 5.17% &#177; 0.001 (SEM; n=3 biological replicates, 4 experimental replicates) were EpCAM+. In mice, all the EpCAM+ cells were immunoreactive to pan-cytokeratin, as determined by IF microscopy. Results were confirmed using Krt1-14;mTmG transgenic mice, with low (8.6 native GFP+ cells per 106 cells analyzed; 0.085% of viable cells), but significant numbers (p &#60; 0.0005) of GFP+ cells present in normal murine BM, that were not the result of randomness compared with multiple negative controls. Further, EpCAM+ cells in mouse blood were more heterogeneous than CD45+ cells (0.58% in BM; 0.13% in blood). These observations conclude that cells expressing cytokeratin proteins are reproducibly detectable among mononuclear cells from human and murine blood and BM. We demonstrate a method of tissue harvesting, flow cytometry, and immunostaining that can be used to identify and determine the function of these pan-cytokeratin epithelial cells in healthy individuals.

This paper presents a reproducible method with new findings on the presence of epithelial cells in normal human and mouse blood and bone marrow using flow cytometry and immunofluorescence microscopy. Krt1-14;mTmG transgenic mice were used as an in vivo method to confirm these findings.

EpCAMおよびサイトケラチンが正常なヒトおよびマウスの血液および骨髄において上皮細胞を発現する証拠

Epithelial cells have been identified in the blood and bone marrow of patients with cancer and other diseases. However, the presence of normal epithelial ...

FluoZin-3アッセイによる哺乳類細胞における亜鉛輸送メカニズムの調査

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

著者スポットライト:ZnT1機能による細胞亜鉛制御の探索 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

In Vitro亜鉛輸送アッセイを用いた哺乳類亜鉛トランスポーターの特性評価(英語)

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...