Name: mutagenesis and analysis of genetic mutations in the gc-rich kiss1 receptor sequence identified in humans with reproductive disorders
Uploaded: 2011-09-04T14:00:00
Description: 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

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 ...

<ol>
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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 ...

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