Produktionen av rekombinanta biofarmaceutiska läkemedel, exempelvis monoklonala antikroppar, är en oumbärlig men ansträngande process. Under 2019 var sju av de tio mest sålda läkemedlen globalt baserade på rekombinant producerade monoklonala antikroppar. En betydande flaskhals i utvecklingen av stabila cellinjer är screening och selektion av högproducerande singelcellkloner. Tekniker som utspädningskloning (limiting dilution) och fluorescensaktiverad cellsortering (FACS) når idag medelmåttiga resultat som bäst, men saknar förmåga att både isolera produktiva singelcellkloner och hålla cellviabilitet på en acceptabel nivå. Samtidigt kan CellCelectorn™, ett helt automatiserat instrument utformat för att detektera, selektera och isolera singelcellkloner, screena celler för att kartlägga deras produktivitet. CellCelectorn™ är utrustad både med brightfield- och fluorescerande kameror och kan ranka samtliga cellkloner i en provplatta efter produktivitet för att sedan överföra de mest lovande klonerna till en destinationsplatta för vidare analyser och singelklonexpansion. Med avstamp i detta instrument var syftet med projektet att utveckla ett automatiserat arbetsflöde för screening och selektion av högproducerande singelcellkloner med hög genomströmning vid generering av stabila cellinjer. Inledande tester med redan utvecklade och produktiva cellinjer genomfördes för att undersöka CellCelectorns™ prestanda och för att upprätta och optimera sekundära parametrar relevanta för instrumentets kapacitet. Då dessa inledande tester var framgångsrika utfördes en polyklonal selektion i syfte att utveckla tre stabila cellinjer producerandes olika antikroppar. Dessa cellinjer skulle sedan användas för att undersöka CellCelectorns™ förmåga att isolera och överföra de mest lovande singelklonerna till en destinationsplatta. Dessa test kunde tyvärr inte genomföras, då kontaminering i de polyklonala poolerna hade uppstått, men de övergripande resultaten av detta projekt indikerar att CellCelectorn™ är kapabel till att screena för och ranka singelkloner efter deras produktivitet samt att selektera högproducerande klonkandidater under en enda arbetsvecka. Framtida tester är dock nödvändiga för att säkerställa CellCelectorns™ förmåga att isolera singelcellkloner med hög genomsnittlig cellviabilitet för att kunna genomföra singelklonexpansion. / The production of recombinant biopharmaceuticals, such as monoclonal antibodies (mAbs), from stable mammalian cell lines is an indispensable yet strenuous process. In 2019, seven of the top ten most sold drugs globally were based on monoclonal antibodies produced recombinantly. A prominent bottleneck in stable cell line development is the screening and selection of high target protein producing single clone candidates. Today, techniques such as limiting dilution and Fluorescence Activated Cell Sorting (FACS) receive moderate success at best at isolating single clones while keeping cell viability high. Simultaneously, the CellCelector™ is a fully automated instrument designed for the detection, selection, and isolation of single cell clones. Containing both a brightfield and fluorescence-detecting camera, the CellCelector™ can screen cells to measure their productivity and rank them accordingly, meaning high target protein producing clones can be selected and transferred to a destination plate for single clone expansion. Thus, this project aimed at developing an automated high-throughput workflow using the CellCelector™ to streamline the screening and selection steps of stable mammalian cell line generation. Several tests with stable proof-of-concept cells were performed to evaluate the performance of the CellCelector™ and to establish favorable secondary parameter settings. As initial proof-of-concept tests showed promise, a polyclonal selection to obtain three stable Human Embryonic Kidney (HEK) cultures expressing different antibodies was carried out in order to test the CellCelector™ proficiency in clone selection and picking. Unfortunately, no clone picking or single clone expansion could be executed due to bacterial contamination in the cell cultures. Nevertheless, the overall results of this project indicate high potential of the CellCelector™ to detect and identify promising stable clone candidates for single clone expansion over the course of a single work week. Future tests are however required to solidify CellCelector™ ability to isolate monoclonal clones while preserving cell viability for single clone expansion.

Ziel dieser Arbeit war die elektrophysiologische Charakterisierung des vor kurzem erstmals von NIESLER et al. (2003) klonierten humanen 5-HT3A+E-Rezeptors. Da dieser Rezeptor-Subtyp ausschließlich im Gastrointestinaltrakt exprimiert wird, ist ein Einfluss auf Nausea und Emesis sehr wahrscheinlich. Es stellt sich demnach die Frage, ob funktionelle Unterschiede zum homomeren 5-HT3A-Rezeptor und zum heteromeren 5-HT3A+B-Rezeptor bestehen, und ob auf molekularer Ebene unterschiedliche Wirkungen emetogener bzw. antiemetischer Pharmaka festzustellen sind. Um die Wirkmechanismen und die Interaktionen eines Pharmakons mit den 5-HT3-Rezeptor-Subtypen beurteilen zu können, erfordert dies genaue Kenntnisse über das biophysikalische Verhalten und die pharmakologische Sensitivität der 5-HT3-Rezeptor-Untereinheiten. Die Experimente erfolgten in-vitro an heterolog in HEK293-Zellen exprimierten Rezeptoren, wobei alleinig die 5-HT3A-Untereinheit in der Lage ist, funktionelle homopentamere Rezeptoren auszubilden. Die 5-HT3E- und 5-HT3B-Untereinheiten können nur zusammen mit der 5-HT3A-Untereinheit an die Zelloberfläche exprimiert werden und funktionelle heteropentamere Rezeptoren bilden. Im Verlauf der Untersuchungen hat sich herausgestellt, dass bei der Transfektion die 5-HT3E- und die 5-HT3B-Untereinheiten im Verhältnis zur 5-HT3A-Untereinheit signifikant schwächer exprimiert werden. Mittels der experimentellen Methode der Patch-Clamp Technik im „excised-patch“ („outside-out“)- und im Ganzzell-Modus war es möglich, die biophysikalischen und pharmakologischen Eigenschaften des heteromeren 5-HT3A+E-Rezeptors im Vergleich mit dem homomeren 5-HT3A-Rezeptor und dem heteromeren 5-HT3A+B-Rezeptor zu analysieren. Bei den Experimenten zur Grundcharakterisierung des humanen 5-HT3A+E-Rezeptor-Subtyps zeigte die Agonisten-Konzentrations-Wirkungskurve mit einem Hill-Koeffizienten von 1,0 einen deutlichen flacheren Verlauf als die Kurve des 5-HT3A-Rezeptor-Subtyps, die einen Hill-Koeffizienten von 1,5 aufwies. Dies spricht für eine geringe Agonisten-Bindungskooperativität des 5-HT3A+E-Rezeptors. Kein Unterschied zeigte sich allerdings in der Affinität zu 5-HT, da die EC50-Werte von beiden Rezeptor-Subtypen im Bereich von ca. 7 µM lagen. Aus dem biphasischen Verlauf der Kurve konnte der Rückschluss gezogen werden, dass bei der Transfektion des heteromeren 5-HT3A+E-Rezeptors der homomere 5-HT3A-Rezeptor parallel exprimiert wird. Dasselbe Verhalten wurde auch schon für den heteromeren 5-HT3A+B-Rezeptor beschrieben (WALSTAB et al. 2008). Bei der Charakterisierung eines heteromeren Rezeptor-Subtyps ergibt sich dadurch die Schwierigkeit, dessen Eigenschaften nicht eindeutig von denen des homomeren Rezeptors unterscheiden zu können. Des Weiteren konnte im Vergleich zum homomeren 5-HT3A-Rezeptor eine schnellere Desensibilisierungszeitkonstante des heteromeren 5-HT3A+E-Rezeptors nachgewiesen werden. Insgesamt deuten die beschriebenen Ergebnisse auf eine erhöhte Sensitivität des Rezeptors für Serotonin hin. Da der 5-HT3A+E-Rezeptor ausschließlich im Gastrointestinaltrakt exprimiert wird, könnte dies ein Hinweis auf eine Beteiligung dieses Rezeptors bei der Vermittlung von Emesis sein. Bei der pharmakologischen Charakterisierung wurden der partielle 5-HT3-Rezeptoragonist Tryptamin, der volle 5-HT3-Rezeptorantagonisten Tropisetron sowie die partiellen 5-HT3-Rezeptorantagonisten Metoclopramid, Tubocurarin, Mirtazapin und der Cannabinoid-Rezeptoragonist Anandamid, welcher eine emetogene Wirkung aufweist, untersucht. Auffällig war ein deutlich flacherer Verlauf der Konzentrations-Wirkungskurve von Metoclopramid (5-HT3A+E-Rezeptor: Hill-Koeffizient = -0,8; 5-HT3A-Rezeptor: Hill-Koeffizient = -1,2) und von Mirtazapin (5-HT3A+E-Rezeptor: Hill-Koeffizient = -0,9; 5-HT3A-Rezeptor: Hill-Koeffizient = -1,3) am heteromeren 5-HT3A+E-Rezeptor. Des Weiteren konnte für Mirtazapin am 5-HT3A+E-Rezeptor ein IC50-Wert von 8,4 nM im Vergleich zu 25,4 nM am 5-HT3A-Rezeptor festgestellt werden. Diese deutlich höhere Potenz von Mirtazapin am untersuchten heteromeren Rezeptor-Subtyp sowie die geringere Bindungskooperativität von Mirtazapin und Metoclopramid am 5-HT3A+E-Rezeptor, stellen einen interessanten Ansatz für eine effektive Pharmakotherapie gastrointestinaler Erkrankungen dar. Die Ergebnisse dieser Arbeit zeigen erstmalig auf molekularer Ebene, die elektrophysiologischen Eigenschaften der humanen 5-HT3A+E-Rezeptoren sowie deren Beeinflussung durch emetogenen und antiemetische Pharmaka. Aufgrund der schwachen Expression der 5-HT3E-Untereinheit gilt es in Zukunft durch einen alternativen Weg der Transfektion, die Effizienz der Ausbeute von 5-HT3A+E-Rezeptoren zu erhöhen. / The aim of this doctor thesis was the electrophysiological characterization of the human 5-HT3A+E receptor which was recently cloned for the first time by NIESLER et al. (2003). Since the expression of this receptor subtype takes place exclusively in gastrointestinal tract, an influence on nausea and emesis is very likely. The question is if functional differences exist between homomeric 5-HT3A receptors and heteromeric 5-HT3A+B receptors, and whether different effects from emetic and antiemetic drugs can be detected at the molecular level. To assess the mechanisms and the interactions of a drug with the 5-HT3 receptor subtypes, knowledge of the biophysical characteristics and the pharmacological sensitivity of the 5-HT3 receptor subunit is required. The experiments were developed in-vitro on heterologous expressed receptors in HEK293-cells, whereat only the 5-HT3A subunit is able to form functional homopentameric receptors. The 5-HT3E and the 5-HT3B subunit can only be expressed on the cell surface and build functional heteropentameric receptors in combination with the 5-HT3A subunit. In the course of the investigations it became obvious that during transfection the 5-HT3E subunit and the 5-HT3B subunit are significantly lesser expressed than the 5-HT3A subunit. Using the patch-clamp technique in the excised-patch (outside-out) and whole-cell configuration it was possible to analyse the pharmacological and biophysical properties of the heteromeric 5-HT3A+E receptor compared with the homomeric 5-HT3A-receptor and the heteromeric 5-HT3A+B receptor. During the characterisation of the human 5-HT3A+E receptor subtype, the agonist concentration-response curve with the hillslope of 1,0 showed a significant flatter course than the graph of the 5-HT3A receptor subtype with a hillslope of 1,5. This indicates a diminished agonist binding-cooperativeness of the 5-HT3A+E receptor. No difference could be detected in the affinity to 5-HT, since the EC50 values of both receptor-subtypes were at the range of 7 µM. The biphasic course of the graph showed that by transfection of the heteromeric 5-HT3A+E receptor the homomeric 5-HT3A-receptor is expressed parallel. The same properties were described also for the 5-HT3A+B receptor (WALSTAB et al. 2008). Therefore it is difficult to distinguish the properties of a homomeric receptor by characterisation of a heteromeric receptor subtype. Furthermore, a faster desensitization of the heteromeric 5-HT3A+E-receptor could be demonstrated in comparison to homomeric 5-HT3A-receptor. Overall, the results described above indicate an increased sensitivity to the receptor for serotonin. As the 5-HT3A+E receptor is expressed exclusively in the gastro-intestinal tract, this could be an indication of involvement of this receptor in the mediation of emesis. During the pharmacological characterisation the partial 5-HT3 receptor agonist tryptamine, the full 5-HT3 receptor antagonist tropisetron as well as the partial 5-HT3 receptor antagonists metoclopramide, tubocurarin, mirtazapin and the cannabinoid receptor agonist anandamide, which has an emetic effect, were examined. The agonist concentration-response curve of metoclopramide (5-HT3A+E receptor: hillslope = -0,8; 5-HT3A receptor: hillslope = -1,2) and of mirtazapin (5-HT3A+E receptor: hillslope = -0,9; 5-HT3A receptor: hillslope = -1,3) showed a significant flatter course at the 5-HT3A+E receptor. Mirtazapin has an IC50 value of 8,4 nM at the 5-HT3A+E receptor in comparison to 25,4 nM at the 5-HT3A receptor. This significant higher potency of mirtazapin at the heteromeric 5-HT3 receptor subtype and the decreased binding-cooperativeness of mirtazapin and meteclopramide at the 5-HT3A+E receptor represent interesting approaches for an effective pharmacotherapy for gastrointestinal diseases. For the first time the results of this thesis showed the electrophysiological properties of the human 5-HT3A+E receptors and their interference by emetic and antiemetic drugs on the molecular level. Due to the decreased expression of 5-HT3E subunit, the goal for the future is to find an alternative way of transfection which increases the rate of yield for the 5-HT3A+E receptors.

Natural killer or NK cells are immunocytes that mediate innate immunity against pathogens and tumors without pre-exposition to the antigen. They are holding rapid antiviral defense during the initial phase of immune response, before starting the production of antibodies and the development of specific cytotoxic T -lymphocytes. On the surface of NK cells is expressed wide range of inhibition and activation receptors. Important family of those receptors are C - type lectin like from which the family of NKR - P1 ("natural killer cell receptor - protein 1") was discovered first. Diploma thesis deals with the preparation/study of mice NK cell activation receptor NKR- P1C and searching for its binding partner. The soluble form of the protein NKR-P1C was prepared by recombinant expression using the transient transfection of HEK293 cell line (human embryonic kidney 293) with wild type or homogenous glycosylation as IgG - Fc fusion protein, from which was it possible to obtain pure dimer of NKR P1C, after process of affinity purification, TEV protease cleavage and HPLC chromatography. The fusion protein was bound to protein A labeled with a fluorescent probe DyLight 488. Mice tissues and cell lines were labeled by this complex for purpose of seeking ligand.

The Natural Killer (NK) cells have an important role in the nonspecific immunity of the or- ganism. They have the ability to identify and to kill tumor cells and cells infected by a virus without preceding sensitization by antigen. Their function is directed by the amount of sti- mulation and inhibition receptors interacting with ligands on the tumor or infected cell. This thesis focuses on the preparation and the study of the complex of rat NK cellular inhi- bition receptor NKR-P1B ("natural killer cell receptor - protein 1B") and its ligand Clrb ("C-type lectin-related ligand b"). The Clrb initiates the inhibition of NKR-P1B, meaning that if the cell express Clrb, it won't be destroyed. If the cell gets infected by the rat cytome- galovirus, it loses Clrb from its surface and its destruction is therefore no longer prevented. Cells infected with this virus defend themselves from destruction by expression of the viral gene of C-type lectin RCTL, which is a homolog of Clrb. Transient transfection of human embryonic kidney 293 cell line with simple glycosylation (HEK293S GnTI− ) was used for the recombinant preparation of the soluble form of these two receptors of the rat NK cells. The native forms of the receptors - disulfidic homo- dimers - were prepared as the fusion construct with IgG Fc (using...

NK buňky (z angl. natural killer cells, přirozeně zabíječské buňky) hrají klíčovou roli při rozpoznávání a ničení nádorových, infikovaných nebo jinak pozměněných buněk. Na svém povrchu nemají antigenně specifické receptory, proto je řadíme mezi složky přirozené imunity. K rozpoznání cílových buněk slouží řada jiných povrchových receptorů. Inhibiční receptory zajišťují buněčnou toleranci, naopak aktivační receptory spouští cytotoxické mechanismy vedoucí k apoptóze a tedy lýzi buňky. Díky této vlastnosti jsou NK buňky intenzivně studovány v souvislosti s imunoterapií nádorových onemocnění. Jedním z aktivačních receptorů je NKp80 rozpoznávající svůj ligand AICL. Oba proteiny patří do rodiny receptorů podobných lektinům C-typu. Tento komplex se účastní nejenom přímé lýze maligních buněk myeloidního charakteru, ale má také důležitou roli v imunomodulaci zánětu. Předmětem této diplomové práce je příprava receptoru NKp80 a jeho ligandu AICL. Receptor NKp80 byl připraven v linii lidských embryonálních ledvinných buněk (HEK 293S GnTI- ). Byly připraveny stabilně transfekované linie produkující protein NKp80 konstitutivně nebo indukovatelně. Zapojení disulfidických můstků a obsazení N-glykosylačních míst proteinu NKp80 bylo ověřeno hmotnostní spektrometrií. Dále byl optimalizován postup Mgr. Jiřího Nového na...

La mort cellulaire programmée ou apoptose est un mécanisme conservé chez les eucaryotes multicellulaires qui contribue au développement embryonnaire et à l'homéostasie cellulaire des organismes. Dans les cellules vivantes, l'activité des protéases qui exécutent le programme de mort cellulaire, les caspases, est contrôlée par des signaux de survie provenant de l'environnement cellulaire. Les caspases initiatrices de l'apoptose régulée par l'environnement, la caspase 9 et la caspase 8 sont activées respectivement par l'apoptosome et par les récepteurs de mort. Les signaux environnementaux, parmi lesquels le contact avec la matrice extracellulaire ou la présence de facteurs de croissance, activent des voies de signalisation contrôlant la machinerie de mort cellulaire. La voie des MAPK1/3 est une voie de signalisation contrôlée par le proto-oncogènes Ras et comportant les kinases Raf, MEK1/2 et MAPK1/3 (ERK2/1 ou p42/p44). La voie des MAPK1/3, qui est impliquée dans la prolifération et la différentiation cellulaire, joue un rôle essentiel dans la survie cellulaire. L'objectif de cette thèse a été de caractériser les mécanismes moléculaires impliqués dans le contrôle de la mort cellulaire par la voie des MAPK1/3. Ce travail est basé sur l'utilisation d'une forme active et inductible de la kinase Raf-1 (DRaf-1:ER) dont l'activation forte et prolongée correspond à une induction pathologique de la voie des MAPK1/3. Nous avons montré que, selon le type cellulaire, l'activation de deltaRaf-1:ER favorise la survie ou la mort cellulaire. Dans les cellules fibroblastiques CCL39, l'activation de deltaRaf-1:ER protège de la mort cellulaire mitochondriale induite par la privation en sérum du milieu de culture. Dans ces conditions, nous avons montré que la stimulation de Raf-1 :ER bloque l'activation de la caspase-9 mais n'empêche pas la délocalisation du cytochrome c, la multimérisation d'APAF1 ni le recrutement de la procaspase 9 dans l'apoptosome. Ce mécanisme post mitochondrial de protection contre la mort cellulaire dépend de la néo-synthèse des protéines et nécessite une activité continue de la kinase MEK. A l'inverse, dans les cellules HEK 293 issues de rein embryonnaire et présentant des caractéristiques neuronales, nous avons montré que l'activation soutenue de la voie des MAPK1/3 par DRaf1-ER induit une mort cellulaire massive. Celle-ci est caractérisée par l'activation des caspases et la fragmentation de l'ADN. La mort cellulaire est détectée plus de 24 heures après l'activation de Raf1-ER, elle est maximale à 48h. L'induction de la mort cellulaire ne requière la synthèse protéique que durant la phase précoce d'activation mais nécessite l'activité continue du module MEK/MAPK. La mort cellulaire résulte de l'activation de la caspase 8 et n'implique pas la voie mitochondriale, elle est caractérisée par une vacuolisation importante du cytoplasme des cellules qui l'apparente à une forme particulière d'apoptose. L'inactivation des fonctions du récepteur fas et de son adaptateur FADD indique que le processus d'activation de la caspase 8 est indépendant de la voie des récepteurs de mort. L'ensemble de ces travaux apporte des connaissances nouvelles sur le contrôle de la mort cellulaire par la voie Raf/MAPK1/3. Nous avons montré que la voie de signalisation peut, selon le contexte cellulaire, favoriser la survie cellulaire ou induire la mort. Dans les deux cas, le contrôle de la mort cellulaire dépend à la fois de la synthèse protéique et de mécanismes post-traductionnels. Les mécanismes moléculaires affectés par l'activation prolongée des MAPK1/3 seraient impliqués aussi bien dans la résistance des cellules tumorales aux traitements proapoptotiques que dans le développement des maladies neurodégénératives.

Fatty acyl-Coenzyme As are metabolites of lipid anabolism and catabolism. A method was developed for their quantitation in extracts of cultured mammalian cells using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Palmitoyl-CoA (C16:0-CoA) is utilized for de novo sphingolipid biosynthesis catalyzed by serine palmitoyltransferase (SPT), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. After reduction to form sphinganine (Sa), dihydroceramide synthase (CerS) can N-acylate the Sa using a second fatty acyl-CoA molecule, forming dihydroceramide (DHCer). The CerS enzyme family utilizes different acyl chain lengths of fatty acyl-CoAs in an isoform-specific manner, resulting in DHCer with N-acyl chains ranging from C16 to C26 [and even longer] in mammalian tissues. DHCer is trans-4,5-desaturated to yield ceramide, which is further metabolized by the addition of moieties at the 1-O-position, forming sphingomyelin (SM) and ceramide monohexose (CMH). The rates of fatty acyl-CoA and sphingolipid biosynthesis were determined using stable isotope-labeling and LC-ESI-MS/MS analysis of the analyte isotopologues and isotopomers. Isotopic labeling of palmitoyl-CoA with [U-13C]-palmitate in HEK293 and RAW264.7 cells was robust and rapid (~ 60% labeling of the metabolite pool in 3 hr). Isotopic labeling of sphingolipids indicated utilization of [M + 16]-palmitoyl-CoA by SPT and CerS isoforms in both cell types. Metabolic flux modeling was applied to the data for [U-13C]-palmitate activation to [M + 16]-palmitoyl-CoA and its subsequent utilization in de novo sphingolipid biosynthesis, and this analysis indicated rapid turn-over rates for palmitoyl-CoA and ceramide in both cell types. Palmitate treatment of cultured cells alters their metabolic status and gene expression, therefore labeling of palmitoyl-CoA by treatment with [1-13C]-acetate was employed. A distribution of mass-shifted palmitoyl-CoA species (isotopologues) is observed based on the number of incorporations of [1-13C]-acetate during de novo biosynthesis, requiring computational analysis to derive two parameters: the isotopic enrichment of the precursor pool, and the fraction of palmitoyl-CoA that was biosynthesized during the experiment. Previous reports by others describe mass isotopomer distribution analysis (MIDA) and isotopomer spectral analysis (ISA) for this purpose, and both calculation approaches indicated concurrent results. In summary, the quantitation of fatty acyl-CoAs and their isotopic enrichment during stable isotope-labeling studies of lipid metabolism can provide data that significantly change the interpretation of analyte quantitation in these experiments, as demonstrated here for investigations of de novo sphingolipid biosynthesis.

Arginine vasopressin (AVP) is a hypothalamic nonapeptide which regulates the hypothalamic-pituitary-adrenal axis response to stress by stimulating the secretion of adrenocorticotropin (ACTH) from corticotroph cells of the anterior pituitary. This effect is mediated by binding of AVP to the pituitary vasopressin receptor (V1bR). The V1bR belongs to the G protein-coupled receptor (GPCR) super family. Repeated stimulation of anterior pituitary cells with AVP has been shown to produce a loss of responsiveness to subsequent AVP stimulation. This phenomenon appears to be mediated by desensitisation of the V1bR, and may be due to phosphorylation of the receptor by G protein-coupled receptor kinase 5 (GRK5). The aim of this research was to establish and validate methods that would allow the role of GRK5 in the desensitisation of V1bR to AVP stimulation to be investigated. As no isoform specific inhibitors for GRK5 were available, HEK293 cells transiently transfected with the rat V1bR were used as a model system for this research. This allowed RNA interference (RNAi) to be used to knockdown GRK5 expression. The protocol for RNAi-mediated knockdown of GRK5 was established as part of this research. Protocols for Western blotting and qRT-PCR were also established to allow the RNAi-mediated knockdown of GRK5 protein and mRNA to be measured. Transfection of HEK293 cells with 10nM GRK5-targeting small interfering RNAs (siRNAs) reduced the expression of GRK5 protein to 53.4% ± 3.4% (mean ± SEM) of that seen in untreated control cells at 84 hours after transfection, while GRK5 mRNA levels were reduced to 28.7% ± 1.9% (mean ± SEM) of that of control cells 48 hours after transfection. An experimental protocol was designed in this research that would coordinate the RNAi-mediated knockdown of GRK5 with transient transfection of the HEK293 cells with the rV1bR. Since, activated V1bRs couple to Gq/11 and stimulate the production of inositol phosphates (IPs), the responsiveness of the V1bR can be determined by measuring the accumulation of [H³]-IPs in cells labelled with [H³]-myo-inositol. In the protocol designed, the effect of GRK5 knockdown on V1bR desensitisation is determined by stimulating HEK293 cells expressing the rV1bR (and previously transfected with GRK5-targeting siRNA) with 0nM or 100nM AVP for 0, 5, 15, 30 or 60 minutes, and comparing the accumulation if IPs over time with that of cells that are not transfected with GRK5-targeting siRNA. This protocol can be used in future to investigate the role of GRK5 in V1bR desensitisation, and may be adapted to determine if other GRK isoforms are involved in V1bR desensitisation.

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No description available.