transgene expression r26 (Worthington Biochemical)

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Collagenase Type 1

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The original balance of enzymatic activities Each lot assayed for collagenase caseinase clostripain and tryptic activities Suggested for epithelial liver lung and adrenal primary cell isolations A dialyzed lyophilized powder

Catalog Number:

ls004194

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100 mg

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Clostridium histolyticum

Cas Number:

9001.12.1

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Structured Review

Worthington Biochemical transgene expression r26

A single-vector tetracycline construct allows doxycycline-regulated expression . (A) Overview showing the regulator plasmid containing the <t>EF1α-tTA</t> cassette (pEntL1L3-EF1α-tTA, left) and the responder plasmid containing the <t>TetO-mCh-Rs1</t> cassette (pEntR3L2 TetO-mCh-Rs1, right). The mCherry and Rs1 cistrons are separated by a P2A ribosomal skip sequence to allow simultaneous expression of both peptides. The entry plasmids were recombined using Gateway technology into the desired destination vector containing the AttR1 and AttR2 Gateway sites. The TetO and EF1α-tTA portions are in opposite orientation (indicated by upside-down text) to minimize steric hindrance between the two promoters, as well as potential cross-activation of the TetO by the EF1α promoter. In addition, flanking insulator sequences are included to minimize any read-through activation of the constructs by surrounding promoters (such as Rosa26) that may lead to

(transgene expression allowed) demonstrate doxycycline-dependent mCherry expression. (D) Schematic of targeted Rosa26 locus and Southern screening strategy. The Rosa26 locus in E14 ES cells was targeted by homologous recombination with the Exp-R26(EF1α-tTA/TetO-mCh-Rs1) construct. Regions in hatch marks indicate the 5' and 3' homology regions of the targeting vector and the endogenous Rosa26 locus (abbreviated R26 in the figure). The location of the 5' recombination Southern probe and HindIII restriction sites are indicated. (E) Southern blots of genomic DNA digested with HindIII and probed as in (D). Heterozygous ES cells at the Rosa26 locus are indicated by the two bands. " width='250' height="auto" />
The original balance of enzymatic activities Each lot assayed for collagenase caseinase clostripain and tryptic activities Suggested for epithelial liver lung and adrenal primary cell isolations A dialyzed lyophilized powder
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1) Product Images from "Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice"

Article Title: Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice

Journal: Stem Cell Research & Therapy

doi: 10.1186/scrt52

A single-vector tetracycline construct allows doxycycline-regulated expression . (A) Overview showing the regulator plasmid containing the EF1α-tTA cassette (pEntL1L3-EF1α-tTA, left) and the responder plasmid containing the TetO-mCh-Rs1 cassette (pEntR3L2 TetO-mCh-Rs1, right). The mCherry and Rs1 cistrons are separated by a P2A ribosomal skip sequence to allow simultaneous expression of both peptides. The entry plasmids were recombined using Gateway technology into the desired destination vector containing the AttR1 and AttR2 Gateway sites. The TetO and EF1α-tTA portions are in opposite orientation (indicated by upside-down text) to minimize steric hindrance between the two promoters, as well as potential cross-activation of the TetO by the EF1α promoter. In addition, flanking insulator sequences are included to minimize any read-through activation of the constructs by surrounding promoters (such as Rosa26) that may lead to

Figure Legend Snippet: A single-vector tetracycline construct allows doxycycline-regulated expression . (A) Overview showing the regulator plasmid containing the EF1α-tTA cassette (pEntL1L3-EF1α-tTA, left) and the responder plasmid containing the TetO-mCh-Rs1 cassette (pEntR3L2 TetO-mCh-Rs1, right). The mCherry and Rs1 cistrons are separated by a P2A ribosomal skip sequence to allow simultaneous expression of both peptides. The entry plasmids were recombined using Gateway technology into the desired destination vector containing the AttR1 and AttR2 Gateway sites. The TetO and EF1α-tTA portions are in opposite orientation (indicated by upside-down text) to minimize steric hindrance between the two promoters, as well as potential cross-activation of the TetO by the EF1α promoter. In addition, flanking insulator sequences are included to minimize any read-through activation of the constructs by surrounding promoters (such as Rosa26) that may lead to "leakiness" or steric interference from endogenous promoter activity. (B, C) HEK-293 cells carrying the Exp-pcDNA3.2(EF1α-tTA/TetO-mCh-Rs1) expression cassette and cultured in doxycycline (suppressed expression) or in the absence of doxycycline (transgene expression allowed) demonstrate doxycycline-dependent mCherry expression. (D) Schematic of targeted Rosa26 locus and Southern screening strategy. The Rosa26 locus in E14 ES cells was targeted by homologous recombination with the Exp-R26(EF1α-tTA/TetO-mCh-Rs1) construct. Regions in hatch marks indicate the 5' and 3' homology regions of the targeting vector and the endogenous Rosa26 locus (abbreviated R26 in the figure). The location of the 5' recombination Southern probe and HindIII restriction sites are indicated. (E) Southern blots of genomic DNA digested with HindIII and probed as in (D). Heterozygous ES cells at the Rosa26 locus are indicated by the two bands.

Techniques Used: Plasmid Preparation, Construct, Expressing, Sequencing, Activation Assay, Activity Assay, Cell Culture, Homologous Recombination

A single copy of the EF1α-tTA regulator region weakly drives expression of a TetO transgene in mice . (A) Areal bone mineral density by DEXA of nine-week-old mice shows that the ColI(2.3)-tTA x R26(EF1α-tTA/TetO-mCh-Rs1) mice have increased bone mass. N = 9 WT, 5 R26(EF1α-tTA/TetO-mCh-Rs1), and 9 ColI(2.3)-tTA x R26(EF1α-tTA/TetO-mCh-Rs1) mice. ***, P

Figure Legend Snippet: A single copy of the EF1α-tTA regulator region weakly drives expression of a TetO transgene in mice . (A) Areal bone mineral density by DEXA of nine-week-old mice shows that the ColI(2.3)-tTA x R26(EF1α-tTA/TetO-mCh-Rs1) mice have increased bone mass. N = 9 WT, 5 R26(EF1α-tTA/TetO-mCh-Rs1), and 9 ColI(2.3)-tTA x R26(EF1α-tTA/TetO-mCh-Rs1) mice. ***, P

Techniques Used: Expressing, Mouse Assay

2) Product Images from "An Upstream Activator Sequence Regulates the Murine Pgk-1 Promoter and Binds Multiple Nuclear Proteins"

Article Title: An Upstream Activator Sequence Regulates the Murine Pgk-1 Promoter and Binds Multiple Nuclear Proteins

Journal: Gene Expression

doi:

Diagram of the Pgk-1 promoter. The solid line represents the DNA sequence upstream of the Pgk-1 ). The cross-hatched bars labeled ES, EA, and AS are the probes used for EMSA and DNasel footprinting experiments. The slashed, black, and gray boxes labeled R1, R2, and R3 refer to the regions within the UAS that bind nuclear proteins. The black box labeled R2 was defined by both DNase1 footprinting and EMSA whereas the slashed and gray boxes labeled R1 and R3 were shown to bind protein by EMSA only; no footprints were obtained over these regions. The locations of R1 and R3 were established by EMSA with oligonucleotide probes or with DNA fragments from the promoter region. The distribution of nuclear DNA binding factors is indicated below the promoter diagram where the intensity of the signal is roughly proportional to the size of the bars.

Figure Legend Snippet: Diagram of the Pgk-1 promoter. The solid line represents the DNA sequence upstream of the Pgk-1 ). The cross-hatched bars labeled ES, EA, and AS are the probes used for EMSA and DNasel footprinting experiments. The slashed, black, and gray boxes labeled R1, R2, and R3 refer to the regions within the UAS that bind nuclear proteins. The black box labeled R2 was defined by both DNase1 footprinting and EMSA whereas the slashed and gray boxes labeled R1 and R3 were shown to bind protein by EMSA only; no footprints were obtained over these regions. The locations of R1 and R3 were established by EMSA with oligonucleotide probes or with DNA fragments from the promoter region. The distribution of nuclear DNA binding factors is indicated below the promoter diagram where the intensity of the signal is roughly proportional to the size of the bars.

Techniques Used: Sequencing, Labeling, Footprinting, Binding Assay

3) Product Images from "Human embryonic stem cells which express hrGFP in the undifferentiated state and during dopaminergic differentiation"

Article Title: Human embryonic stem cells which express hrGFP in the undifferentiated state and during dopaminergic differentiation

Journal: Restorative neurology and neuroscience

doi: 10.3233/RNN-2009-0521

Characterization of cellular phenotypes present in BGO1V2-EFG cells after 18 days of differentiation following removal from the PA6 feeder cells. (A) MAP2; (B) β III -tubulin; (C) GFAP; (D) SMA; (E, F) nestin; (G, H) TH. Staining for all markers

Figure Legend Snippet: Characterization of cellular phenotypes present in BGO1V2-EFG cells after 18 days of differentiation following removal from the PA6 feeder cells. (A) MAP2; (B) β III -tubulin; (C) GFAP; (D) SMA; (E, F) nestin; (G, H) TH. Staining for all markers

Techniques Used: Staining

4) Product Images from "Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice"

Article Title: Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice

Journal: Stem Cell Research & Therapy

doi: 10.1186/scrt52

R26(EF1α-tTA/TetO-mCh-Rs1) function in mouse ES cells . (A) FACS analysis showing doxycycline-inducible mCherry expression in E14 mouse cells carrying the Exp-R26(EF1α-tTA/TetO-mCh-Rs1) construct. (B) Induction of Rs1 expression and treatment with the agonist RS67333 results in increased cAMP accumulation in mouse ES cells. Both basal and ligand-induced increases in cAMP are detectable. In addition, serotonin does not induce increased cAMP accumulation in either the wildtype or Rs1-expressing cells. (C) Schematic showing the differentiation protocol for making suspension EBs. (D) Expression and ligand activation of Rs1 during EB formation results in larger EB size. (E) Quantitation of EB size in the different culture conditions using ImageJ. A minimum of 114 EBs were measured for each condition. The analysis was performed on three separate EB differentiation experiments with similar results. Error bars represent average +/- 1 SD.

Figure Legend Snippet: R26(EF1α-tTA/TetO-mCh-Rs1) function in mouse ES cells . (A) FACS analysis showing doxycycline-inducible mCherry expression in E14 mouse cells carrying the Exp-R26(EF1α-tTA/TetO-mCh-Rs1) construct. (B) Induction of Rs1 expression and treatment with the agonist RS67333 results in increased cAMP accumulation in mouse ES cells. Both basal and ligand-induced increases in cAMP are detectable. In addition, serotonin does not induce increased cAMP accumulation in either the wildtype or Rs1-expressing cells. (C) Schematic showing the differentiation protocol for making suspension EBs. (D) Expression and ligand activation of Rs1 during EB formation results in larger EB size. (E) Quantitation of EB size in the different culture conditions using ImageJ. A minimum of 114 EBs were measured for each condition. The analysis was performed on three separate EB differentiation experiments with similar results. Error bars represent average +/- 1 SD.

Techniques Used: FACS, Expressing, Construct, Activation Assay, Quantitation Assay

5) Product Images from "Extramedullary erythropoiesis in the adult liver requires BMP4/Smad5 dependent signaling"

Article Title: Extramedullary erythropoiesis in the adult liver requires BMP4/Smad5 dependent signaling

Journal: Experimental hematology

doi: 10.1016/j.exphem.2009.01.004

Figure Legend Snippet: Analysis of BFU-E in the bone marrow and liver during the recovery from PHZ induced anemia

Techniques Used:

6) Product Images from "Angiogenic factor signaling regulates centrosome duplication in endothelial cells of developing blood vessels"

Article Title: Angiogenic factor signaling regulates centrosome duplication in endothelial cells of developing blood vessels

Journal: Blood

doi: 10.1182/blood-2010-01-266197

Flt-1 −/− mutant endothelial cells from developing vessels display aneuploidy and chromosome aberrations . Endothelial cells from WT and flt-1 −/− ES cell–derived vessels were isolated via magnetic bead isolation and analyzed for chromosome number and abnormalities. (A-B) Giemsa-stained WT (A) and flt-1 −/− (B) endothelial cell metaphase spreads with 40 and 47 chromosomes (CS), respectively ( Mus musculus 2n = 40). Arrows in panel B point to abnormal triradial chromosome configurations. (C) Scatter plot showing chromosome number in WT versus flt-1 −/− endothelial cells from developing vessels. Each dot represents one cell; red dots, cells with 40 CS; and blue dots, aneuploid cells. WT, n = 25; flt-1 −/− , n = 25. * P ≤ .05.

Figure Legend Snippet: Flt-1 −/− mutant endothelial cells from developing vessels display aneuploidy and chromosome aberrations . Endothelial cells from WT and flt-1 −/− ES cell–derived vessels were isolated via magnetic bead isolation and analyzed for chromosome number and abnormalities. (A-B) Giemsa-stained WT (A) and flt-1 −/− (B) endothelial cell metaphase spreads with 40 and 47 chromosomes (CS), respectively ( Mus musculus 2n = 40). Arrows in panel B point to abnormal triradial chromosome configurations. (C) Scatter plot showing chromosome number in WT versus flt-1 −/− endothelial cells from developing vessels. Each dot represents one cell; red dots, cells with 40 CS; and blue dots, aneuploid cells. WT, n = 25; flt-1 −/− , n = 25. * P ≤ .05.

Techniques Used: Mutagenesis, Derivative Assay, Isolation, Staining

High VEGF signaling leads to an increased frequency of excess centrosomes in endothelial cells of developing vessels . (A-B) Day 8 ES cell–derived WT and flt-1 −/− mutant vessels were stained for PECAM-1 (red); note the vessel overgrowth and dysmorphogenesis in panel B. (C-J) Endothelial cells of WT and flt-1 −/− mutant vessels were analyzed for centrosome numbers. (C-D) Day 8 ES cell cultures were fixed and stained for γ-tubulin (red), PECAM-1 (green), and DRAQ5 nuclear dye (blue). (F-G) Day 8 ES cell cultures that were WT or flt-1 −/− and carried a PECAM-H2B::GFP transgene (green) were dissociated and attached to tissue culture dishes before fixation and staining for γ-tubulin (red). (E) Percentage of PECAM-H2B::GFP-positive cells with more than 2 centrosomes (WT, n = 159; flt-1 −/− , n = 336). (I-J) WT and flt-1 −/− embryos were harvested at E9.5, and yolk sacs were stained for γ-tubulin (red), PECAM-1 (green), and DRAQ5 nuclear dye (blue). (H) Percentage of PECAM-positive yolk sac endothelial cells with more than 2 centrosomes (WT, n = 88; flt-1 −/− , n = 180). (L-M) HUVECs were incubated for 96 hours in low or high VEGF and then stained for γ-tubulin (red) and DRAQ5 nuclear dye (blue). (K) Percentage of HUVECs with more than 2 centrosomes (low VEGF, n = 2393; high VEGF, n = 3011). Arrows point to areas of cells with more than 2 centrosomes, and arrowheads point to areas of cells with 1 or 2 centrosomes. (D,I-J,M) Insets: Centrosomes at higher magnification. (A-B) Scale bar represents 50 μm. (C-M) Scale bar represents 5 μm. All experiments were performed at least 3 times. * P

Figure Legend Snippet: High VEGF signaling leads to an increased frequency of excess centrosomes in endothelial cells of developing vessels . (A-B) Day 8 ES cell–derived WT and flt-1 −/− mutant vessels were stained for PECAM-1 (red); note the vessel overgrowth and dysmorphogenesis in panel B. (C-J) Endothelial cells of WT and flt-1 −/− mutant vessels were analyzed for centrosome numbers. (C-D) Day 8 ES cell cultures were fixed and stained for γ-tubulin (red), PECAM-1 (green), and DRAQ5 nuclear dye (blue). (F-G) Day 8 ES cell cultures that were WT or flt-1 −/− and carried a PECAM-H2B::GFP transgene (green) were dissociated and attached to tissue culture dishes before fixation and staining for γ-tubulin (red). (E) Percentage of PECAM-H2B::GFP-positive cells with more than 2 centrosomes (WT, n = 159; flt-1 −/− , n = 336). (I-J) WT and flt-1 −/− embryos were harvested at E9.5, and yolk sacs were stained for γ-tubulin (red), PECAM-1 (green), and DRAQ5 nuclear dye (blue). (H) Percentage of PECAM-positive yolk sac endothelial cells with more than 2 centrosomes (WT, n = 88; flt-1 −/− , n = 180). (L-M) HUVECs were incubated for 96 hours in low or high VEGF and then stained for γ-tubulin (red) and DRAQ5 nuclear dye (blue). (K) Percentage of HUVECs with more than 2 centrosomes (low VEGF, n = 2393; high VEGF, n = 3011). Arrows point to areas of cells with more than 2 centrosomes, and arrowheads point to areas of cells with 1 or 2 centrosomes. (D,I-J,M) Insets: Centrosomes at higher magnification. (A-B) Scale bar represents 50 μm. (C-M) Scale bar represents 5 μm. All experiments were performed at least 3 times. * P

Techniques Used: Derivative Assay, Mutagenesis, Staining, Incubation

7) Product Images from "Speed and segmentation control mechanisms characterized in rhythmically-active circuits created from spinal neurons produced from genetically-tagged embryonic stem cells"

Article Title: Speed and segmentation control mechanisms characterized in rhythmically-active circuits created from spinal neurons produced from genetically-tagged embryonic stem cells

Journal: eLife

doi: 10.7554/eLife.21540

Neurosphere differentiation and composition. ( A and B ) 10 days-post ES cell differentiation neurospheres were dissociated and cell composition was quantified using FACS. Plots of V3, V2a, and V1 interneurons and motor neurons from Sim1:Cre;R26/C:LSL:Tomato, Chx10:Cre;R26/C:LSL:Tomato, En1:Cre;R26/C:LSL:Tomato, and Hb9:GFP ES cell reporter lines, respectively are shown after differentiation with ( A ) 1000 nM and ( B ) 5 nM SAG. ( C ) Two Chx:10:Cre;R26/C:LSL:Tomato ES cell lines were differentiated 10 days under a range of SAG concentrations. Tomato+V2a neuron number was quantified using FACS. ( D ) Chx:10:Cre;R26/C:LSL:Tomato lines #1 and #2 were differentiated with 15 nM and 40 nM SAG, respectively. 5 μM DAPT was applied days 6–10. Standardized tomato + V2a neurons relative to no DAPT control. DOI: http://dx.doi.org/10.7554/eLife.21540.004

Figure Legend Snippet: Neurosphere differentiation and composition. ( A and B ) 10 days-post ES cell differentiation neurospheres were dissociated and cell composition was quantified using FACS. Plots of V3, V2a, and V1 interneurons and motor neurons from Sim1:Cre;R26/C:LSL:Tomato, Chx10:Cre;R26/C:LSL:Tomato, En1:Cre;R26/C:LSL:Tomato, and Hb9:GFP ES cell reporter lines, respectively are shown after differentiation with ( A ) 1000 nM and ( B ) 5 nM SAG. ( C ) Two Chx:10:Cre;R26/C:LSL:Tomato ES cell lines were differentiated 10 days under a range of SAG concentrations. Tomato+V2a neuron number was quantified using FACS. ( D ) Chx:10:Cre;R26/C:LSL:Tomato lines #1 and #2 were differentiated with 15 nM and 40 nM SAG, respectively. 5 μM DAPT was applied days 6–10. Standardized tomato + V2a neurons relative to no DAPT control. DOI: http://dx.doi.org/10.7554/eLife.21540.004

Techniques Used: Cell Differentiation, FACS

Incubation:

Article Title: Endothelial MT1‐ MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis
Article Snippet: .. Briefly, after fat removal under a microscope, aortas were incubated for 5 min at 37°C in collagenase solution (collagenase type I, 3.33 mg/ml, Worthington, LS004194), thus allowing removal of the adventitia with forceps. .. The aortas were then cut into small pieces (1–2 mm), and a cell suspension was obtained by incubation for 45 min at 37°C in 6 mg/ml type I collagenase and 2.5 mg/ml elastase (Worthington, LS002290) diluted in DMEM.

Gradient Centrifugation:

Article Title: Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion
Article Snippet: .. Following further digestion with 10mg/mL type 1 collagenase (Worthington) and DNase 1, the suspensions were passed through a 100 μm sieve and separated by density gradient centrifugation using Histopaque (Sigma-Aldrich, MO, USA). .. DMSCs were cultured in α-MEM medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific, MA, USA), penicillin/streptomycin (100 U/mL and 100 mg/mL, respectively, Life Technologies), and 2mM L-glutamine (Sigma-Aldrich).