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

चार्ल्स एच. हूड फाउंडेशन युवा अन्वेषक बाल स्वास्थ्य अनुसंधान पुरस्कार (बोस्टन, MA) - इस काम आंशिक रूप से नेशनल इंस्टीट्यूट ऑफ बाल स्वास्थ्य और मानव विकास (HD059015 R21 NICHD) के प्रजनन शाखा द्वारा वित्त पोषित किया गया था.

1. साइट - निर्देशित अत्यधिक जीसी - अमीर KISS1R जीन अनुक्रम के mutagenesis <ol><li> साँचा: एक एन टर्मिनस से इनकार Myc टैग के साथ मानव KISS1R का पूरा सीडीएनए अनुक्रम. इस अनुक्रम pCS2 + अभिव्यक्ति वेक्टर, जो बाद transfections के लिए इस्तेमाल...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPR54+regulates+ERK1/2+activity+and+hypothalamic+gene+expression+in+a+Galpha(q/11)+and+beta-arrestin-dependent+manner.">GPR54 regulates ERK1/2 activity and hypothalamic gene expression in a Galpha(q/11) and beta-arrestin-dependent manner.</a> PLoS One. 5, e12964-e12964 (2010).</li><li>Bianco, S. D. C., Vandepas, L., Correa-Medina, M., Gereben, B., Mukherjee, A., Kuohung, W., Carroll, R., Teles, M. G., Latronico, A. C., Kaiser, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KISS1R+Intracellular+Trafficking+and+Degradation:+Effect+of+the+Arg386Pro+Disease-Associated+Mutation.">KISS1R Intracellular Trafficking and Degradation: Effect of the Arg386Pro Disease-Associated Mutation.</a> Endocrinology. , (2011).</li><li>Hutchison, C. 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साइट - निर्देशित mutagenesis पर तीन दशकों के लिए nucleotide परिवर्तन लक्षित जीन की कोडन अनुक्रम में शुरू करने से प्रोटीन समारोह का अध्ययन करने के लिए इस्तेमाल किया गया है. मूल तकनीक 1978 में ब्रिटिश कनाडा रसायनज्ञ और नो...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td></tr><tr><td> 10x PCRx बढ़ाने समाधान </td><td> Invitrogen </td><td> 52391 </td></tr><tr><td> PfuUltra उच्च विश्वस्तता डीएनए पोलीमरेज़ वैकल्पिक डिटर्जेंट </td><td> Stratagene </td><td> 600385 </td></tr><tr><td> DPN मैं </td><td> न्यू इंग्लैंड Biolabs </td><td> R0176 </td></tr><tr><td> XL10 गोल्ड Ultracompetent ई. कोलाई कोशिकाओं </td><td> Stratagene </td><td> 200314 </td></tr><tr><td> DMEM </td><td> Cellgro </td><td> 10-013-CV </td></tr><tr><td> भ्रूण गोजातीय सीरम </td><td> अटलांटा बायोलॉजिकल </td><td> S11550 </td></tr><tr><td> Geneporter अभिकर्मक अभिकर्मक </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 अवरोध करनेवाला कॉकटेल और PMSF </td><td> सांता क्रूज़ जैव प्रौद्योगिकी </td><td> SC - 24,948 </td></tr><tr><td> पियर्स बीसीए प्रोटीन परख किट </td><td> थर्मो वैज्ञानिक </td><td> 23225 </td></tr><tr><td> विरोधी Myc टैग (4A6 क्लोन) agarose संयुग्म </td><td> Millipore </td><td> 16-219 </td></tr><tr><td> 2x लोड हो रहा है बफर </td><td> BioRad </td><td> 161-0737 </td></tr><tr><td> मानदंड Tris - एचसीएल मिल में बना हुआ जेल, 4-15% ढाल </td><td> BioRad </td><td> 345-0028 </td></tr><tr><td> Immobilon-FL PVDF झिल्ली </td><td> Millipore </td><td> IPFL00010 </td></tr><tr><td> ओडिसी अवरुद्ध बफर </td><td> लाइट - भ्रष्टाचार बायोसाइंसेज </td><td> 927-40000 </td></tr><tr><td> 10xTBS </td><td> BioRad </td><td> 170-6435 </td></tr><tr><td> खरगोश प्रतिरक्षी विरोधी-myc </td><td> सेल संकेतन </td><td> 2272 </td></tr><tr><td> विरोधी खरगोश बकरी IRDye 800CW </td><td> लाइट - भ्रष्टाचार बायोसाइंसेज </td><td> 926-32211 </td></tr><tr><td> ओडिसी इमेजिंग इन्फ्रारेड सिस्टम </td><td> लाइट - भ्रष्टाचार बायोसाइंसेज </td><td></td></tr><tr><td> हॉल्ट protease अवरोध करनेवाला कॉकटेल (100x) </td><td> थर्मो वैज्ञानिक </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

Kisspeptin रिसेप्टर (KISS1R) में उत्परिवर्तनों रोगियों में प्रजनन विकारों के साथ जुड़े रहे हैं. यहाँ हम का वर्णन कैसे KISS1R के जीसी अमीर अनुक्रम में के रूप में अच्छी तरह से KISS1R constructs का उपयोग immunoprecipitation और पश्चिमी धब्बा द्वारा रिसेप्टर की गिरावट मार्ग विशेषताएँ के रूप में ब्याज की म्यूटेशनों शुरू करने के लिए.

Mutagenesis और जीसी अमीर KISS1 रिसेप्टर प्रजनन विकार के साथ मनुष्य में पहचाने गए अनुक्रम में आनुवंशिक परिवर्तन के विश्लेषण

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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