trpm7  (Alomone Labs)

Journal: Frontiers in Cellular Neuroscience

Article Title: Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes

doi: 10.3389/fncel.2018.00319

Figure Lengend Snippet: MOR and TREK-1 co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (*** P

Article Snippet: Primary antibody used are as follow: chicken anti-GFAP (1:500, ab5541, Millipore), rabbit anti-TREK-1 (1:100, APC-047, Alomone Labs), and rabbit anti-MOR (1:200, sc-15310, Santa Cruz Biotechnology).

Techniques: Staining, Labeling

Journal: Frontiers in Cellular Neuroscience

Article Title: Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes

doi: 10.3389/fncel.2018.00319

Figure Lengend Snippet: Astrocytic glutamate release upon MOR activation is mediated by TREK-1. (A) Left, schematic diagram of sniffer-patch with acutely dissociated astrocytes from pSicoR-TREK-1-shRNA-mCherry-injected hippocampus and GluR1LY-GFP-expressing HEK cells. Right, differential interference contrast image and fluorescent image of GluR1LY-GFP-expressing HEK cell (green) and TREK-1-shRNA-mCherry-infected hippocampal astrocyte (red). (B) Representative traces of Ca 2+ response and inward current in acutely dissociated hippocampal astrocyte of scrambled-shRNA and TREK-1-shRNA-injected mouse. (C) Summary bar graph for DAMGO-induced glutamate current normalized by the full activation current in scrambled shRNA and TREK-1-shRNA-injected mice. Data from at least three independent mice for each group. Unpaired two-tailed t -test with Welch’s correction (* P

Article Snippet: Primary antibody used are as follow: chicken anti-GFAP (1:500, ab5541, Millipore), rabbit anti-TREK-1 (1:100, APC-047, Alomone Labs), and rabbit anti-MOR (1:200, sc-15310, Santa Cruz Biotechnology).

Techniques: Activation Assay, shRNA, Injection, Expressing, Infection, Mouse Assay, Two Tailed Test

Journal: Frontiers in Cellular Neuroscience

Article Title: Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes

doi: 10.3389/fncel.2018.00319

Figure Lengend Snippet: MOR and TREK-1 are co-expressed in hippocampal astrocytes of MOR-mCherry mouse. (A) A structured illumination microscopic (SIM) image of a hippocampal astrocyte of MOR-mCherry mouse, immunostained with antibodies against GFAP and TREK-1. (B) 2D histogram of mCherry intensity and TREK-1 intensity in the SIM image (A) . Pearson’s coefficient (r) is calculated within the colocalized pixels which were automatically thresholded by Imaris 9.2 program. (C) Representative confocal images of co-localization of MOR-mCherry and TREK-1 in astrocytes in hippocampal cornu ammonis 1 (CA1) of MOR-mCherry mice. (D) An example of ROI generation within a single astrocyte. (E) Representative 2D histogram of mCherry intensity and TREK-1 intensity in the confocal image. (F) Pearson’s coefficients (r) between mCherry and TREK-1 from confocal images of single astrocytes. (G) A confocal image of hippocampal tissues from WT and TREK-1 KO mice, stained with an antibody against TREK-1.

Article Snippet: Primary antibody used are as follow: chicken anti-GFAP (1:500, ab5541, Millipore), rabbit anti-TREK-1 (1:100, APC-047, Alomone Labs), and rabbit anti-MOR (1:200, sc-15310, Santa Cruz Biotechnology).

Techniques: Mouse Assay, Staining

Journal: PLoS ONE

Article Title: Modulation of Human Cardiac TRPM7 Current by Extracellular Acidic pH Depends upon Extracellular Concentrations of Divalent Cations

doi: 10.1371/journal.pone.0170923

Figure Lengend Snippet: pH o -dependent potentiation and inhibition of the TRPM7 current in human atrial cardiomyocytes. (A) Pooled data of the effects of extracellular acidification when perfusing with physiological solution containing divalent cations. (B) Pooled data of the effects when perfusing with extracellular divalent-free medium. All cells in (A) and (B) were dialysed with [Mg] i -free internal solution. Currents at positive (+80 mV; unfilled circles) and negative (-120 mV; filled circles) potentials are expressed relative to those measured pH o 7.4 (taken as 100%). Each data point represents the mean ± SEM from 5 to 26 cells. Significant differences are denoted as *P

Article Snippet: Images of labeled TRPM7 proteins using a rabbit polyclonal TRPM7 antibody from Alomone (catalog number: ACC-047).

Techniques: Inhibition

Journal: PLoS ONE

Article Title: Modulation of Human Cardiac TRPM7 Current by Extracellular Acidic pH Depends upon Extracellular Concentrations of Divalent Cations

doi: 10.1371/journal.pone.0170923

Figure Lengend Snippet: Effect of extracellular pH on the TRPM7 current in physiological and nominally divalent-free medium. (A-B) Currents recorded at +80 mV and –120 mV from two different human atrial cardiomyocytes before and during acidification of extracellular solutions either in the presence (A) or in the absence of extracellular divalent cations (B). Periods of exposure to acidic pH and nominally divalent-free solutions are indicated by horizontal bars. (C-D) Current-voltage relationships under steady-state conditions obtained using voltage ramps from +80 mV to –120 mV in the same cells as in A and B, respectively. (Insets in C-D) Ratios between TRPM7 currents recorded under various pH o conditions over the current at pH 7.4. Breaks were made in the graph for potentials were currents at pH 7.4 were close to 0, making the ratio inaccurate. Notice that both the inward and outward currents are potentiated by acidic pH when perfusing with extracellular solution containing divalent cations, whereas they were reduced in nominally divalent-free medium.

Article Snippet: Images of labeled TRPM7 proteins using a rabbit polyclonal TRPM7 antibody from Alomone (catalog number: ACC-047).

Techniques:

Journal: PLoS ONE

Article Title: Modulation of Human Cardiac TRPM7 Current by Extracellular Acidic pH Depends upon Extracellular Concentrations of Divalent Cations

doi: 10.1371/journal.pone.0170923

Figure Lengend Snippet: Comparison of the response to acidic pH o between cardiomyocytes from patients with vs without previous myocardial ischemia. (A-B) Effects of acidic pH o on TRPM7 current density measured in steady-state in extracellular solution either containing Ca 2+ and Mg 2+ (A) or nominally free of the divalent (B). Internal free-[Mg 2+ ] i = ~0 mM. Measurements taken at +80 mV and –120 mV. Each data point represents the mean ± SEM in cardiomyocytes obtained from patients with coronary artery disease ( filled symbols ), when superfusing with divalent cations (n cells = 8–23) or without divalents (n cells = 2–14), and in cells obtained from patients without history of coronary disease ( unfilled symbols) , when superfusing with divalent cations (n cells = 5–8), and without divalent cations (n cells = 3–5). Statistically significant differences are denoted as *P

Article Snippet: Images of labeled TRPM7 proteins using a rabbit polyclonal TRPM7 antibody from Alomone (catalog number: ACC-047).

Techniques:

Journal: PLoS ONE

Article Title: Modulation of Human Cardiac TRPM7 Current by Extracellular Acidic pH Depends upon Extracellular Concentrations of Divalent Cations

doi: 10.1371/journal.pone.0170923

Figure Lengend Snippet: Effect of acidic external pH on the TRPM7 current in human cardiomyocyte under physiological conditions. (A-B) Time diaries of whole-cell currents measured at +80 mV and -120 mV (A) and current-voltage relationships (B) in the same cell, when dialyzed with internal solution containing 5.5 mM MgCl 2 (calculated free [Mg 2+ ] i ~0.7 mM) and perfused initially with 1.8-mM [Ca 2+ ] o , 0.9-mM [Mg 2+ ] o extracellular solutions at pH 7.4 and thereafter at pH 6.0, pH 5.0, and pH 4.0. Periods of exposure to 7.2 mM Mg 2+ , to acidic pH o , and to nominally divalent-free solutions are indicated by horizontal bars.

Article Snippet: Images of labeled TRPM7 proteins using a rabbit polyclonal TRPM7 antibody from Alomone (catalog number: ACC-047).

Techniques:

Journal: PLoS ONE

Article Title: Modulation of Human Cardiac TRPM7 Current by Extracellular Acidic pH Depends upon Extracellular Concentrations of Divalent Cations

doi: 10.1371/journal.pone.0170923

Figure Lengend Snippet: Detection of TRPM7 in human atrial cardiomyocytes. (A-B) Time diaries of whole-cell TRPM7 currents measured at +80 mV and –120 mV in cells dialyzed with either Mg 2+ -free internal solution (A) or with 0.7 mM [Mg 2+ ] i (B). Periods of exposure to 7.2 mM Mg 2+ and nominally divalent-free extracellular solutions are indicated by horizontal bars. Insets: Traces from the same cells showing current voltage-relations obtained using voltage ramps from +80 mV to –120 mV. (C) Images of labeled TRPM7 proteins. Atrial cardiomyocytes were incubated with (a, b, c) or without (negative control; a’, b’, c’) TRPM7 primary antibody, and co-stained with Hoechst 33342 (for the nucleus; blue ), Phalloidin-Alexa Fluor 546 (for F-actin cytoskeleton; red ), goat anti-mouse Alexa Fluor 488 (for TRPM7; green ). Uppermost row: image of F-actin staining. Middle row: image of TRPM staining. Lowermost row: merged image.

Article Snippet: Images of labeled TRPM7 proteins using a rabbit polyclonal TRPM7 antibody from Alomone (catalog number: ACC-047).

Techniques: Labeling, Incubation, Negative Control, Staining

Journal: PLoS ONE

Article Title: Modulation of Human Cardiac TRPM7 Current by Extracellular Acidic pH Depends upon Extracellular Concentrations of Divalent Cations

doi: 10.1371/journal.pone.0170923

Figure Lengend Snippet: Variability of the TRPM7 current in human atrial cardiomyocytes from patients with AF. (A-B) Traces of the TRPM7 currents measured at +80 mV and –120 mV from two different human atrial cardiomyocytes, one from a patient with (A), the other from a patient without (B) history of myocardial ischemia, respectively. (C-D) Steady-state current-voltage relations obtained using voltage ramps from +80 mV to –120 mV in the same cells as in (A-B), respectively. (Insets C-D): Current-voltage relations of the typical Mg 2+ -sensitive current displaying steep outward rectification and E rev ~0 mV. Periods of block of TRPM7 current with 7.2 mM Mg 2+ , 100 μM Gd 3+ or 100 μM 2-APB are indicated by horizontal bars.

Article Snippet: Images of labeled TRPM7 proteins using a rabbit polyclonal TRPM7 antibody from Alomone (catalog number: ACC-047).

Techniques: Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity

doi: 10.3390/ijms21217897

Figure Lengend Snippet: Possible association of zinc with neuronal death after pilocarpine-induced SE. This schematic drawing represents several chain reactions that may occur after carvacrol and 2-APB treatment in pilocarpine-induced SE. ( A ) These are the possible cellular pathways through which neuronal death occurs after pilocarpine-induced SE. ( B ) Blocking TRPM7 by carvacrol and 2-APB can inhibit several chain reactions that are thought to occur following pilocarpine-induced SE.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-TRPM7 (diluted 1:400; Alomone labs, Jerusalem, Israel), mouse andti-4HNE (diluted 1:500; Alpha Diagnostic Intl.

Techniques: Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity

doi: 10.3390/ijms21217897

Figure Lengend Snippet: 2-APB treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 5–6 from each seizure group). * p

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-TRPM7 (diluted 1:400; Alomone labs, Jerusalem, Israel), mouse andti-4HNE (diluted 1:500; Alpha Diagnostic Intl.

Techniques: Over Expression, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity

doi: 10.3390/ijms21217897

Figure Lengend Snippet: Carvacrol treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 7 from each seizure group). * p

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-TRPM7 (diluted 1:400; Alomone labs, Jerusalem, Israel), mouse andti-4HNE (diluted 1:500; Alpha Diagnostic Intl.

Techniques: Over Expression, Immunofluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

doi: 10.3389/fncel.2016.00013

Figure Lengend Snippet: Genetic deletion of TWIK-1 and TREK-1 genes together does not alter the electrophysiological properties of astrocytes. (A,B) Bar graph summary of the V M and R in from WT and TWIK-1 −/− /TREK-1 −/− astrocytes. (C) Representative whole-cell current profiles from WT and TWIK-1 −/− /TREK-1 −/− astrocytes, respectively. (D) Averaged I-V plots from these two genotypes, where the whole-cell current amplitudes in both inward and outward directions were comparable. (E) The RI values were also comparable between the two genotypes.

Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

doi: 10.3389/fncel.2016.00013

Figure Lengend Snippet: Quinine does not reveal the functional contribution of TWIK-1 and TREK-1 in single or double gene knockout mice. (A) Representative astrocyte V M recordings first in 100 μM BaCl 2 bath application for 5 min, followed by addition of 400 μM quinine for 20 min, from a WT, TREK-1 −/− , TWIK-1 −/− and TWIK-1 −/− /TREK-1 −/− astrocyte as indicated in situ . (B) Summary of 400 μM quinine-induced V M depolarization (Δ V M 1) and the total V M depolarization induced by BaCl 2 plus quinine from all four genotypes. (C) Representative whole-cell current recordings first in aCSF as control, then in 100 μM BaCl 2 plus 400 μM quinine, and washout. Representative I–V relationships were shown in the right panel. (D) Summary of RI values from four genotypes obtained from astrocyte recordings in the presence of 400 μM quinine and 100 μM BaCl 2 together in bath. The RI values were comparable among the four genotypes.

Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

Techniques: Functional Assay, Double Knockout, Mouse Assay, In Situ

Journal: Frontiers in Cellular Neuroscience

Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

doi: 10.3389/fncel.2016.00013

Figure Lengend Snippet: TREK-1 gene deletion does not alter the electrophysiological properties of astrocytes. (A) Identification of astrocyte in hippocampal slices based on cellular morphology and positive SR101 staining in CA1 region. (B,C) Membrane potential ( V M ), and input resistance ( R in ) in WT and TREK-1 −/− astrocytes. (D) Representative astrocyte whole-cell passive conductance from a WT and a TREK-1 −/− astrocyte are shown separately as indicated. The voltage commands used for whole-cell current induction are displayed on the left panel. (E) I-V plots show the averaged current amplitudes from WT and TREK-1 −/− astrocytes. The equation for rectification index (RI) is also illustrated and the resulted RI values are summarized in (F) .

Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

Techniques: Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

doi: 10.3389/fncel.2016.00013

Figure Lengend Snippet: K ir4.1 inhibition does not reveal the functional contribution of TREK-1 in TREK-1 −/− astrocytes. (A) Representative V M response to K ir 4.1 inhibitor 100 μM BaCl 2 from a WT and a TREK-1 −/− astrocyte, as indicated in situ . Δ V M indicates the peak V M depolarization during a 5 min BaCl 2 bath application. (B) Summary of 100 μM BaCl 2 -induced V M depolarization, where the V M depolarization was comparable between WT and TREK-1 −/− astrocytes. (C) Representative whole-cell current recorded first in control, then 5 min in 100 μM BaCl 2 , and washout. (D) I-V plots derived from recordings in (C) . The Ba 2+ -sensitive currents in I-V plots were obtained from sweep subtraction. The Ba 2+ -sensitive currents were shown in expanded y -axis in the inset that showed a moderate inward rectification in both WT, RI = 0.91, and TREK-1 KO, RI = 0.90, respectively. (E) Summary of RI values from WT and TREK-1 −/− astrocytes obtained from recordings in the presence of 100 μM BaCl 2 for K ir 4.1 inhibition; the RI values were comparable between the two groups.

Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

Techniques: Inhibition, Functional Assay, In Situ, Derivative Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

doi: 10.3389/fncel.2016.00013

Figure Lengend Snippet: TREK-1 channels are predominantly located in cytoplasm. (A) Fractionation western blot results revealed the subcellular distribution of TREK-1 channels in cytoplasmic fraction vs . membrane fraction in two independent tests of mice hippocampal samples. Anti-glial fibrillary acidic protein (GFAP) (50 kDa) and ATP1α2 (112 kDa) were markers for cytoplasmic and membrane fractions, respectively. The blots shown in (A) were first incubated with anti-TREK-1 antibody and then re-probed with the rest of other primary antibodies sequentially after the original membranes were stripped with stripping buffer (see “Materials and Methods” Section). (B) Bar graph summary showing the relative ratio of TREK-1 proteins located in cytoplasmic vs . membrane fractions. Data are shown as mean ± SEM. Numbers indicate the times of observations. ** p

Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

Techniques: Fractionation, Western Blot, Mouse Assay, Incubation, Stripping Membranes

Journal: Frontiers in Cellular Neuroscience

Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

doi: 10.3389/fncel.2016.00013

Figure Lengend Snippet: K ir 4.1 inhibition does not reveal the functional contribution of TWIK-1/TREK-1 in double gene knockout mice. (A) Representative V M response to K ir 4.1 inhibitor, 100 μM BaCl 2 , from a WT and a TWIK-1 −/− /TREK-1 −/− astrocyte as indicated in situ . (B) Summary of 100 μM BaCl 2 -induced V M depolarization, where the V M depolarization was comparable between WT and TWIK-1 −/− /TREK-1 −/− astrocytes. (C) Representative whole-cell current recorded first in control, then 5 min in 100 μM BaCl 2 , and washout. I–V relationships were shown in (D) . (D) I–V plots derived from recordings in (C) . The Ba 2+ -sensitive currents, in I-V plots were obtained from sweep subtraction. The Ba 2+ - sensitive currents were shown in expanded y -axis in the inset that showed a moderate inward rectification in both WT, RI = 0.91, and double gene knockout mice, RI = 0.90, respectively. (E) Summary of RI values from WT and TWIK-1 −/− /TREK-1 −/− astrocytes obtained from recordings in the presence of 100 μM BaCl 2 for K ir 4.1 inhibition; the RI values were comparable between the two groups.

Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

Techniques: Inhibition, Functional Assay, Double Knockout, Mouse Assay, In Situ, Derivative Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

doi: 10.3389/fncel.2016.00013

Figure Lengend Snippet: Expression of astrocyte K + channels in TREK-1, TWIK-1 single, and TWIK-1/TREK-1 double gene knockout mice. (A) Schematic illustrations of genetic deletion of four transmembrane domains, including two pore-forming regions, in TREK-1 knockout (TREK-1 −/− , upper), and TWIK-1 knockout (TWIK-1 −/− , lower) mice. (B) PCR genotyping confirmation of successful genetic knockout of the targeted genes in TWIK-1 −/− , TREK-1 −/− , and TWIK-1/TREK-1 double knockout (TWIK-1 −/− /TREK-1 −/− ) mice. (C) The relative quantity of mRNA of K + channels in freshly dissociated mature astrocytes. The results were examined from wild type (WT), TWIK-1 −/− , TREK- 1−/− , and TWIK-1 −/− /TREK-1 −/− astrocytes using qRT-PCR.

Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

Techniques: Expressing, Double Knockout, Mouse Assay, Knock-Out, Polymerase Chain Reaction, Genotyping Assay, Quantitative RT-PCR