This work was partially funded by the Reproductive Branch of the National Institute of Child Health and Human Development (NICHD &#x2013; R21 HD059015) and by the Charles H. Hood Foundation Young Investigator Child Health Research Award (Boston, MA).

1. 	Site-directed mutagenesis of highly GC-rich KISS1R gene sequence
<ol>
<li>Template: full cDNA sequence of the human KISS1R with a Myc-tag fused to its N-terminus. This sequence is cloned into the pCS2+ expression vector, which is compatible with the mammalian cell lines subsequently used for transfections. This expression vector is referred to herein as pCS2+MycKISS1R.</li>
<li>Primer design: primers are designed to carry desired mutations, according to instructions of the Quikchange Site-Directed Mutagenesis kit (Stratagene). In summary:
<ul>
		<li>Both (forward and reverse) primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid (e.g. forward and reverse primers are complementary to each other) 
<img src="/files/ftp_upload/2897/step1.jpg" alt="Step 1" /></li>
		<li>Primers should be 25 to 45 bases long and end in one or more C or G bases 
				<img src="/files/ftp_upload/2897/step2.jpg" alt="Step 2" /> 
				<img src="/files/ftp_upload/2897/step3.jpg" alt="Step 3" /></li>
		<li>Introduced mutation(s) should be in the middle of primer and flanked by &tilde;10&#x2013;15 bases of correct sequence on both sides</li>
		<li>Melting temperature (Tm) of primers should be equal or greater than 78&deg;C. Use the following formulas to estimate Tm:
 <ul>
				<li>When introducing mutations: Tm = 81.5 + 0.41(%GC) &#x2013; 675/N &#x2013; % mismatch (N is the primer length in bases)</li>
				<li>When introducing insertions or deletions: Tm = 81.5 + 0.41(%GC) &#x2013; 675/N	(N does not include the bases which are being inserted or deleted)</li>
				<li>Use desalted primers (no further purifications necessary). 
					<img src="/files/ftp_upload/2897/step4.jpg" alt="Step 4" /></li>
 </ul>
 </li>
 </ul>
 </li>
 <li>The best amplification conditions include the addition of PCRx Enhancer solution (Invitrogen) and DMSO. At least 2 concentrations of DMSO, such as 4% and 8%, should be tested. PCR conditions are shown in Table I and in Figure 1; and results of a representative PCR amplification of pCS2+MycKISS1R is shown in Figure 2.</li>
 <li>Eliminate parental DNA on amplification products by digestion of methylated DNA with DpnI for 1h 30min at 37&deg;C: mix in a centrifuge tube 17.5&mu;l of PCR product with 2&mu;l of 10x NEBuffer 4 and 0.5&mu;l of DpnI (10U; New England Biolabs).</li>
 <li>Transform DpnI-treated PCR product: mix 2&mu;l of DpnI-treated DNA with 45&mu;l of XL10-Gold Ultracompetent E. coli (Stratagene) in pre-chilled 15 ml cell culture tube. Add 2&mu;l of &beta;-mercaptoethanol and follow Stratagene instructions. Plate 100-400&mu;l of transformed bacteria in LB-agar plates containing 100&mu;g/ml ampicillin and incubate at 37&deg;C.</li>
 <li>Miniprep 2 to 4 individual colonies to isolate plasmid DNA. Confirm the successful introduction of desired mutations by sequencing and analysis of isolated DNA.</li>
</ol>
2. 	Transient transfection of MycKISS1R into HEK-293 cells
<ol>
<li>The following experiments are performed in human embryonic kidney cells (HEK-293) cultured in a CO2 incubator (5% CO2) at 37&deg;C in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.</li>
<li>Seed HEK-293 cells at 2.5x105 cells/ml in 6-well plates and let them grow overnight at 37&deg;C before transfection. NOTE: (i) use triplicate wells for each experimental condition; (ii) ideal cell confluence at the time of transfection is 30%-50%.</li>
<li>Transfect HEK-293 cells using the GenePorter Transfection Reagent (Genlantis), according to the manufacturer's instructions: Mix half of the serum-free DMEM with 0.5&mu;g pCS2+ MycKISS1R plus 0.5&mu;g control (empty) vector to totalize 1&mu;g of DNA/well. Mix the other half with 10&mu;l transfection reagent per &mu;g of DNA transfected. NOTE: Ideal plasmid DNA concentration may vary.</li>
</ol>
3. 	Cell treatment and lysis
<ol>
<li>24h after transfection, replace cell medium with 1ml DMEM containing 2.5% FBS (to decrease cell metabolism). NOTE: This decrease in serum may facilitate and/or amplify the detection of results.</li>
<li>Add lysosome inhibitor (100&mu;g/ well of Leupeptin) directly into each well of one entire 6-well plate. Incubate at 37&deg;C for 6 or 16 h (or other desired times)</li>
<li>Add freshly prepared proteasome inhibitor (10&mu;M/ well of MG132) directly into all wells of two entire 6-well plates. Incubate at 37&deg;C for 2, 4, 6 or 16h (or desired times). Add vehicle to all wells of the fourth 6-well plate (0 time-point) and incubate at 37&deg;C for 16h</li>
<li>When incubation is over, move plates to ice and perform this entire lysis procedure on ice to prevent protein degradation:
<ul>
			<li>To increase protein yield, combine the triplicates on 6-well plates in a single centrifuge tube</li> 
			<li>Aspirate medium and wash cells once with 1ml of ice-cold phosphate buffered saline (PBS)</li>
			<li>Add 100&mu;l of ice-cold lysis buffer (20mM HEPES, pH 7.4, 1% NP-40, 150mM NaCl, 1mM EDTA, 0.25% sodium deoxycholate) containing protease inhibitors (1x cocktail containing 100mM AEBSF-HCl, 80&mu;M aprotinin, 5mM bestatin, 1.5mM E-64, 0.5M EDTA, 2mM leupeptin and 1mM pepstatin A, plus 2mM PMSF) to each well</li>
 	<li>Remove cells with a cell scraper and transfer cell lysates to centrifuge tubes </li>
			<li>Pass cells &tilde;10 times through a 20-gauge needle. NOTE: Do not sonicate samples intended for western blot detection of membrane proteins. Sonication leads to aggregation of membrane proteins, which will not migrate properly during electrophoresis</li>
			<li>Incubate cell lysates for 1h at 4&deg;C on a rocking platform</li>
			<li>Centrifuge cell lysates at 4&deg;C for 10 min at 10,000 x rpm and transfer supernatants to new tubes. NOTE: Do not disturb pellets during this step</li>
			<li>Determine protein concentration in 10&mu;l of supernatants using the BCA method (Pierce)</li>
			<li>Dilute lysates to 1mg/ml with lysis buffer containing protease inhibitors.</li>
</ul>
</li>
</ol>
4. 	Immunoprecipitation and Western blot detection of MycKISS1R
<ol>
<li>Perform the following immunoprecipitation steps on ice (or at 4&deg;C):
<ul>
		<li>Wash the appropriate amount of agarose-conjugated anti-myc antibody (2.5&mu;g/ sample) twice with ice-cold PBS and add this to 400&mu;g of lysate protein.</li>
		<li>Immunoprecipitate the MycKISS1R on lysates overnight at 4&deg;C in a rocking platform with gentle agitation.</li>
		<li>Spin down agarose beads by pulse centrifugation at 4&deg;C (up to 10,000 x rpm)</li> 
		<li>Aspirate and discard supernatants (without disturbing the pellets)</li>
		<li>Wash beads once with ice-cold lysis buffer and twice with ice-cold PBS. Invert tubes gently before spinning</li>
		<li>Ressuspend beads containing antibody-bound MycKISS1R in 2x sample loading buffer containing 10% &beta;-mercaptoethanol.</li>
 </ul>
 </li>
<li>Western blot of MycKISS1R immunocomplexes:
<ul> 
<li>Heat samples for 30 min at 37&deg;C. NOTE: Do not boil samples intended for western blot detection of membrane proteins. Like sonication, boiling also leads to aggregation of these proteins</li>
<li>Move tubes immediately into ice for 5 min</li>
<li>Separate proteins by SDS-PAGE in a 4-15% gradient gel.</li> 
<li>Transfer to Immobilon-FL PVDF membrane (for infra-red detection) at 25V for 30 min in Transfer buffer (48mM Tris base, 39mM glycine, 1.2mM SDS, 20% methanol, pH 9.2) using the Bio-Rad Semi-Dry Transfer Apparatus</li>
<li>Wash membranes for 5 min at room temperature with Tris-Buffered Saline (TBS) and block for 1h at room temperature with Licor Odyssey blocking on a rocking platform (alternatively, 5% milk in TBS can be used to block non-specific binding).</li>
<li>Incubate membranes overnight at 4&deg;C with rabbit anti-myc antibody (1:500) in blocking solution containing 0.1% Tween-20</li> 
<li>Remove primary antibody and wash membranes 3 times of 5 min each with TBS containing 0.1% Tween-20 (TBST)</li>
<li>Incubate membranes for 1h at room temperature with goat Infra-RedDye &reg; 800CW-labelled anti-rabbit IgG (1:10,000) in blocking buffer containing 0.1% Tween-20 and 0.01% SDS</li> 
<li>Remove secondary antibody, wash membranes 3 times of 5 min each with TBST and one last time with TBS only (to remove remaining Tween-20)</li>
</ul>
</li>
<li>Imaging and quantification of MycKISS1R using the LI-COR Odyssey Infra-Red Imager:
<ul> 
<li>The MycKISS1R on the membranes will be imaged using the LI-COR Odyssey Infra-Red Imager. To begin, place membrane on bottom left corner of Odyssey scanner, aligning it with the grid. Cover with the rubber mat, smooth out bubbles with roller and close the lid</li>
<li>Create a new project file on the computer. Name the file, click "done", and then enter the scanner login on "scan". Size the scanner console box to fit your membrane, and then choose 169&mu;m resolution and medium image quality</li> 
<li>Choose intensity settings for 700 (red) and 800 (green) channels according to expected strength of each signal. This is for signal visualization purposes only and will not influence quantification. Click "start scan"</li>
<li>Name and save the scan, then click "OK" to open it on a new window for quantification. MycKISS1R monomers should be visible at approximately 43kD</li>
<li>Using the "box" tool (on the left-hand sidebar), draw a box around the first band. Drag the box around to make sure all bands fit in, then "copy" and "paste" the box over all bands</li>
<li>Select all boxes using "Ctrl+A", and then select the option to subtract median background. Click "report" on the top menu and a spreadsheet with the quantification values will appear. Representative results shown here are represented as fold-increase over untreated cells (time zero)</li></ul></li>
</ol>
5. 	Representative Results:
<ol>
	<li>Site-directed mutagenesis of highly GC-rich KISS1R gene sequence: 
Table 1. shows the combination of reagents to improve efficiency of KISS1R amplification. This combination is been successfully used to introduce several mutations directed against distinct regions of the KISS1R cDNA sequence, as well as for the amplification of this highly GC-rich gene. Figure 1 shows cycling and amplification conditions for the mutagenesis of KISS1R. These conditions have been modified from the QuikChange Site-Directed Mutagenesis kit (Stratagene). 
Figure 2. shows a representative result using this optimized protocol. The addition of 4% or 8% DMSO combined with the PCRx Enhancer significantly improves yield of amplification of the GC-rich KISS1R cDNA-containing plasmid. In this representative experiment, 4% DMSO provided slightly better amplification conditions compared to 8% DMSO. Transformation of DpnI-treated amplification products with ultracompetent E. coli typically yields 100 to over 1,000 colonies, and the rate of successful introduction of desired mutations is 80-90% as determined by DNA sequencing.</li>
<li>Use of MycKISS1R constructs to study receptor physiology: 
 In this representative experiment, the wild type pCS2+MycKISS1R amplified according to the optimized protocol described here is used to study in vivo KISS1 receptor degradation in a relevant cell line (HEK-293) transiently expressing MycKISS1R. After treating the transfected HEK-293 cells with lysosome (leupeptin) or proteasome (MG132) inhibitors according to the protocol described in methods, the cells are lysed and processed for western blot. The top panel of Figure 3 shows the scan of MycKISS1R monomers whereas the bottom panel shows the quantification of the bands shown on top panel. Quantification of bands indicates that neither 6h nor 16h of leupeptin treatment affected MycKISS1R protein levels. Conversely, treatment with MG132 resulted in a time-dependent increase in MycKISS1R protein in these cells, which culminates with a 45-fold accumulation of the receptor after 16h of incubation with MG132. These observations indicate that, unlike most G protein-coupled receptors, KISS1R is degraded by the proteasome (rather than the lysosome).</li>
</ol>

<table border="1">
<tbody>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>DMSO</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Reagents</td>
 <td>0</td>
 <td>4%</td>
 <td>8%</td>
 </tr>
 <tr>
 <td>Water</td>
 <td>35</td>
 <td>33</td>
 <td>31</td>
 </tr>
 <tr>
 <td>10x Pfu Ultra Taq buffer</td>
 <td>5</td>
 <td>5</td>
 <td>5</td>
 </tr>
 <tr>
 <td>dNTP mix (10mM)</td>
 <td>1</td>
 <td>1</td>
 <td>1</td>
 </tr>
 <tr>
 <td>Primer sense (25pmol/&mu;l)</td>
 <td>1</td>
 <td>1</td>
 <td>1</td>
 </tr>
 <tr>
 <td>Primer antisense (25pmol/&mu;l)</td>
 <td>1</td>
 <td>1</td>
 <td>1</td>
 </tr>
 <tr>
 <td>10x PCRx Enhancer Solution</td>
 <td>5</td>
 <td>5</td>
 <td>5</td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>0</td>
 <td>2</td>
 <td>4</td>
 </tr>
 <tr>
 <td>Pfu Ultra Taq polymerase (2.5U/&mu;l)</td>
 <td>1</td>
 <td>1</td>
 <td>1</td>
 </tr>
 <tr>
 <td>plasmid DNA (20ng/&mu;l)</td>
 <td>1</td>
 <td>1</td>
 <td>1</td>
 </tr>
</tbody>
</table>
Table 1. Combination of reagents successfully used to mutate and amplify the GC-rich KISS1R
<img src="/files/ftp_upload/2897/2897fig1.jpg" alt="Figure 1" /> 
Figure 1. Cycling conditions of successful mutagenesis and amplification of GC-rich KISS1R: A 2min hot start was followed by 18 cycles of 30 sec melting at 95&deg;C; 1 min annealing at 55&deg;C and 6 min extension at 68&deg;C. An additional 10 min extension at 68&deg;C was added at the end of the last cycle. These settings were adjusted from the original mutagenesis protocol of the QuikChange II XL-Site-Directed Mutagenesis kit (Stratagene).
<img src="/files/ftp_upload/2897/2897fig2.jpg" alt="Figure 2" /> 
Figure 2. Visualization of GC-rich pCS2+MycKISS1R amplified in the presence or absence of DMSO: Five &mu;l aliquots of pCS2+MycKISS1R amplified in the presence of 0, 4 or 8% DMSO were loaded in this representative 1% agarose gel stained with ethidium bromide and visualized using UV light. The plasmid bands of 6Kb are visible on both lanes loaded with PCR products amplified in the presence of 4% and 8% DMSO, but not on the first lane, which was loaded with a PCR product amplified in the absence of DMSO.
<img src="/files/ftp_upload/2897/2897fig3.jpg" alt="Figure 3" /> 
Figure 3. Effect of leupeptin or MG132 on levels of MycKISS1R protein in HEK-293 cells: HEK-293 cells expressing MycKISS1R were treated with 100&mu;g/ml leupeptin or 10&mu;M MG132 at 37&deg;C for the designated times. MycKISS1R on 400&mu;g of cell lysate was immunoprecipitated with 2.5&mu;g of agarose-conjugated anti-myc antibody and analyzed by western blot. (A) LI-COR Odyssey detection of MycKISS1R after incubation of immunoblots with rabbit anti-Myc-tag antibody followed by incubation with IRDye 800CW-labeled anti-rabbit; (B) Quantification of MycKISS1R bands shown in (A) using the LI-COR Odyssey quantification software. Results are represented as fold-increase over untreated cells (time 0).

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No conflicts of interest declared.

Site-directed mutagenesis has been used to study protein function by introducing nucleotide changes in the coding sequence of targeted genes for over three decades. The original technique was described in 1978 by the British-Canadian Chemist and Nobel Prize winner Michael Smith 10. Michael Smith shared the 1993 Nobel Prize in Chemistry with Kary Mullis, the American Biochemist who invented the Polymerase Chain Reaction (PCR) 11. The original method described by Michael Smith has been improved ove...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>10x PCRx Enhancer Solution</td>
 <td>Invitrogen</td>
 <td>52391</td>
 </tr>
 <tr>
 <td>PfuUltra High-fidelity DNA Polymerase Alternative Detergent</td>
 <td>Stratagene</td>
 <td>600385</td>
 </tr>
 <tr>
 <td>Dpn-I</td>
 <td>New England Biolabs</td>
 <td>R0176</td>
 </tr>
 <tr>
 <td>XL10-Gold Ultracompetent E. coli cells</td>
 <td>Stratagene</td>
 <td>200314</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Cellgro</td>
 <td>10-013-CV</td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Atlanta Biologicals</td>
 <td>S11550</td>
 </tr>
 <tr>
 <td>Geneporter Transfection Reagent</td>
 <td>Genlantis</td>
 <td>T201007</td>
 </tr>
 <tr>
 <td>Leupeptin</td>
 <td>Calbiochem</td>
 <td>108975</td>
 </tr>
 <tr>
 <td>MG132</td>
 <td>Calbiochem</td>
 <td>47491</td>
 </tr>
 <tr>
 <td>10xPBS</td>
 <td>Ambion</td>
 <td>AM9625</td>
 </tr>
 <tr>
 <td>Protease inhibitor cocktail and PMSF</td>
 <td>Santa Cruz Biotechnology</td>
 <td>Sc-24948</td>
 </tr>
 <tr>
 <td>Pierce BCA Protein Assay Kit</td>
 <td>Thermo Scientific</td>
 <td>23225</td>
 </tr>
 <tr>
 <td>anti-Myc tag (clone 4A6) agarose conjugate</td>
 <td>Millipore</td>
 <td>16-219</td>
 </tr>
 <tr>
 <td>2x loading buffer</td>
 <td>BioRad</td>
 <td>161-0737</td>
 </tr>
 <tr>
 <td>Criterion Tris-HCl precast gel, 4-15% gradient</td>
 <td>BioRad</td>
 <td>345-0028</td>
 </tr>
 <tr>
 <td>Immobilon-FL PVDF membrane</td>
 <td>Millipore</td>
 <td>IPFL00010</td>
 </tr>
 <tr>
 <td>Odyssey Blocking Buffer</td>
 <td>LI-COR Biosciences</td>
 <td>927-40000</td>
 </tr>
 <tr>
 <td>10xTBS</td>
 <td>BioRad</td>
 <td>170-6435</td>
 </tr>
 <tr>
 <td>Rabbit anti-myc antibody</td>
 <td>Cell signaling</td>
 <td>2272</td>
 </tr>
 <tr>
 <td>Goat anti-rabbit IRDye 800CW</td>
 <td>LI-COR Biosciences</td>
 <td>926-32211</td>
 </tr>
 <tr>
 <td>Odyssey Imaging Infrared System</td>
 <td>LI-COR Biosciences</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Halt Protease Inhibitor Cocktail (100x)</td>
 <td>Thermo Scientific</td>
 <td>78430</td>
 </tr>
</tbody>
</table>

mutagenesis and analysis of genetic mutations in the gc-rich kiss1 receptor sequence identified in humans with reproductive disorders

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in adulthood 1,2,3. Inactivating mutations in KISS1R identified in patients have been associated with iodiopathic hypogonadotropic hypogonadism (IHH) 1,2 and precocious puberty 4. Functional studies of these mutants are crucial for our understanding of the mechanisms underlying the regulation of reproduction by this receptor as well as those shaping the disease outcomes, which result from abnormal KISS1R signaling and function. However, the highly GC-rich sequence of the KISS1R gene makes it rather difficult to introduce mutations or amplify the gene encoding this receptor by PCR.
Here we describe a method to introduce mutations of interest into this highly GC-rich sequence that has been used successfully to generate over a dozen KISS1R mutants in our laboratory. We have optimized the PCR conditions to facilitate the amplification of a range of KISS1R mutants that include substitutions, deletions or insertions in the KISS1R sequence. The addition of a PCR enhancer solution, as well as of a small percentage of DMSO were especially helpful to improve amplification. This optimized procedure may be useful for other GC-rich templates as well.
The expression vector encoding the KISS1R is been used to characterize signaling and function of this receptor in order to understand how mutations may change KISS1R function and lead to the associated reproductive phenotypes. Accordingly, potential applications of KISS1R mutants generated by site-directed mutagenesis can be illustrated by many studies 1,4,5,6,7,8. As an example, the gain-of-function mutation in the KISS1R (Arg386Pro), which is associated with precocious puberty, has been shown to prolong responsiveness of the receptor to ligand stimulation 4 as well as to alter the rate of degradation of KISS1R 9. Interestingly, our studies indicate that KISS1R is degraded by the proteasome, as opposed to the classic lysosomal degradation described for most G protein-coupled receptors 9. In the example presented here, degradation of the KISS1R is investigated in Human Embryonic Kidney Cells (HEK-293) transiently expressing Myc-tagged KISS1R (MycKISS1R) and treated with proteasome or lysosome inhibitors. Cell lysates are immunoprecipitated using an agarose-conjugated anti-myc antibody followed by western blot analysis. Detection and quantification of MycKISS1R on blots is performed using the LI-COR Odyssey Infrared System. This approach may be useful in the study of the degradation of other proteins of interest as well.

Watch this Scientific Journal Video about Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders at JoVE.com

Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot.

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in ...

mutagenesis-analysis-genetic-mutations-gc-rich-kiss1-receptor

Research

JoVE Journal

Biology

13.9K 조회수.  University of Miami Miller School of Medicine. kisspeptin 수용체 (KISS1R)에 돌연변이가 환자의 생식 장애와 관련된 있습니다. 여기 immunoprecipitation과 서양 얼룩에 의한 수용체의 저하 경로를 특성화 KISS1R의 GC 풍부한 순서뿐만 아니라 KISS1R 구조의 사용으로 관심 변이를 소개하는 방법을 설명합니다

비디오: Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and MET&#916;14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors &#945; and &#946; in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.