pkd1 gene  (Lpath Inc)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modeling Pkd1 gene-targeted strategies for correction of polycystic kidney disease

doi: 10.1016/j.omtm.2023.03.016

Figure Lengend Snippet: Full-length genomic Pkd1 construct introduced into Pkd1 loss-of-function mouse model completely rescues PKD phenotype (A) Schematic representation of full-length Pkd1 gene construct, Pkd1 wt , transferred in the Pkd1 loss-of-function model. Pr, Pkd1 promoter region; ∗RI, marked allele with EcoRI site. (B) Expression of Pkd1 gene in kidneys of Pkd1 −/− mouse with two full-length Pkd1 wt1 and Pkd1 wt2 transgenes by TaqMan qPCR at birth. Pkd1 expression is normalized to Hprt1 and compared with wild-type ( Pkd1 +/+ ) kidneys as fold change. (C) Expression of polycystin-1 (Pc1) protein was carried out by immunoblot in newborn Pkd1 −/− kidneys with Pkd1 wt transfers. Gapdh served as a loading control. Quantitative evaluation of Pc1 expression was normalized to wild-type kidneys ( Pkd1 +/+ ) set as 1. (D) Renal histology of Pkd1 loss of function with Pkd1 wt transfers (lines 1 and 2) is indistinguishable from that of wild-type normal kidneys: P0, birth; P30, 1 month; and 4M, 4 months. Number of mice is indicated in Table S2 . H&E, scale as indicated.

Article Snippet: Pkd1 levels were assessed with the TaqMan master mix and predesigned duplex gene expression assays for Pkd1 , Mm00465434_m1 (FAM-MGB) (cat. no. 4331182), and for Hprt1 , Mm00446968_m1 (VIC-MGB) (cat. no. 4448489) (Applied Biosystems), and Pkd2 levels with PowerUp SYBR Green using the following primers: forward, 5′-CGAGTCAGAAACGGATCCTGCTCCA, and reverse, 5′-CCAGTGACTGCTCCCATTCAGCTC.

Techniques: Construct, Expressing, Western Blot, Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modeling Pkd1 gene-targeted strategies for correction of polycystic kidney disease

doi: 10.1016/j.omtm.2023.03.016

Figure Lengend Snippet: Transgene transfer in Pkd1 loss-of-function mouse with full-length genomic SB Pkd1 suppresses PKD phenotype (A) Full-length Pkd1 gene construct, SB Pkd1 , where “SB” 0.9 kb cassette replaces the Pkd1 promoter. The SB Pkd1 allele is marked by ∗RI. Pkd1 −/− transgene transfers and control mice are color coded below. (B) Pkd1 expression in Pkd1 −/− mouse with one-copy or high-copy SB Pkd1 transfer was analyzed by TaqMan qPCR at birth and normalized to Hprt1 . Levels are indicated as fold change relative to wild-type kidneys. (C) Relative expression levels of Pc1 protein were assessed by immunoblot in Pkd1 −/− kidneys with SB Pkd1 transfers and normalized to wild-type kidneys set as 1. Gapdh served as a loading control. Pc1 quantification is on the right. (D) Longitudinal analysis of 2KW/BW percentage in Pkd1 −/− with SB Pkd1 transfers. Number of mice is indicated in bars. ∗∗∗∗p < 0.0001; ns, non-significant. (E) Cyst measurements (Cyst [%]) over time revealed substantial correction by SB Pkd1 transfers in Pkd1 −/− kidneys. ♦♦♦♦ p < 0.0001; ♦♦ p < 0.01; ns, non-significant. (F) BUN measurement analysis of Pkd1 −/− with SB Pkd1 transfers. ∗∗∗∗p < 0.0001. (G) Longitudinal renal histologic analysis in Pkd1 −/− with one-copy or high-copy SB Pkd1 transfer shows initiation of cysts in cortical region at P3 or in medulla at P10/P15, respectively, and then progression to all renal regions of the kidneys. Number of mice is indicated in Table S2 . H&E, scale as indicated. (H) Representative sections of tubular cyst origin in the parental Pkd1 −/− , Pkd1 −/− with one-copy SB Pkd1 , or high-copy SB Pkd1 transfer. Tubular segments were stained using immunofluorescent markers for proximal (green), distal (blue), and collecting (red) as depicted on the pictogram. Distribution of specific tubular segment diameter was analyzed at selected time points in the histogram (below) with colors matching to the tubular markers. The black histogram accounts for unstained, multistained, and glomerular cysts. Relative frequency of cysts from a specific tubular origin was quantified per total number of tubules of that same segment (below). Cystic diameter threshold was defined at ≥22 μm and the shaded area represents normal tubular size. (I) Global quantitative analysis of cystic tubular segments relative to all tubular segments in Pkd1 −/− with one- or high-copy SB Pkd1 transfers. Renal cysts with one-copy SB Pkd1 are abrogated in proximal tubules, delayed in distal tubules, and incompletely rescued in collecting ducts during maturation. High-copy SB Pkd1 transfer in kidneys of Pkd1 −/− mice protected all tubular segments from developing cysts during maturation and, at post-maturation, was sufficient to suppress proximal and collecting tubular cyst development but insufficient for distal tubules. ♦♦♦♦ p < 0.0001, ♦♦ p < 0.01, ♦ p < 0.05.

Article Snippet: Pkd1 levels were assessed with the TaqMan master mix and predesigned duplex gene expression assays for Pkd1 , Mm00465434_m1 (FAM-MGB) (cat. no. 4331182), and for Hprt1 , Mm00446968_m1 (VIC-MGB) (cat. no. 4448489) (Applied Biosystems), and Pkd2 levels with PowerUp SYBR Green using the following primers: forward, 5′-CGAGTCAGAAACGGATCCTGCTCCA, and reverse, 5′-CCAGTGACTGCTCCCATTCAGCTC.

Techniques: Construct, Control, Expressing, Western Blot, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modeling Pkd1 gene-targeted strategies for correction of polycystic kidney disease

doi: 10.1016/j.omtm.2023.03.016

Figure Lengend Snippet: PKD phenotype of Pkd1 loss-of-function mouse was markedly delayed by Pkd1 Mini (A) Representation of Pkd1 Mini with SB regulatory cassette adjoined to Pkd1 cDNA followed by β-globin sequences (β). Pkd1 −/− with the Pkd1 Mini gene and controls are color coded below. (B) Pkd1 expression in Pkd1 −/− with the Pkd1 Mini was quantified by TaqMan qPCR, normalized to Hprt1 , and compared with wild-type kidneys as fold change. (C) Expression levels of Pc1 protein were evaluated by immunoblot, normalized to Gapdh, and compared with wild-type kidneys set as 1. Relative quantification of Pc1 expression is shown on the right. (D) Longitudinal analysis of 2KW/BW (%) in Pkd1 −/− with the Pkd1 Mini transfer showed drastical increased at P10–P15, in comparison with control Pkd1 −/− and Pkd1 +/+ groups (shared with Figure 2 D). Number of mice is indicated in the bars. ∗∗∗∗p < 0.0001, Pkd1 −/− ♦♦♦♦ p < 0.0001. (E) Cyst measurements (Cyst [%]) over time in Pkd1 −/− mice with Pkd1 Mini transfer were compared with Pkd1 −/− mice (shared with Figure 2 E). ♦♦♦♦ p < 0.0001; ♦♦ p < 0.01; ♦ p < 0.05; ns, non-significant. (F) BUN of Pkd1 −/− with Pkd1 Mini transfer was monitored relative to control groups (shared with Figure 2 F). ∗∗∗∗p < 0.0001, ∗p < 0.05. (G) Histological renal phenotype progression in Pkd1 −/− with Pkd1 Mini transfer exhibits small cysts at birth, with numerous glomerular cysts. Frequency of glomerular cysts was determined as a function of total glomerular and tubular cysts or of total tubules (cystic and non-cystic). Number of mice for histology is indicated in Table S2 . H&E, scale as indicated. (H) Representative sections of tubular cyst origin in parental Pkd1 −/− and in Pkd1 −/− with Pkd1 Mini transfer by immunofluorescence. Histograms with color matching to the tubular markers indicate the relative frequency of cysts from a specific tubular origin per total number of tubules from that same segment (below) at P0, P5, and P10 and in Pkd1 −/− kidneys at P0 (shared with Figure 2 H). Black histogram accounts for unstained, multistained, and glomerular cysts. Cystic diameter threshold was defined at ≥22 μm and shaded area represents normal tubular size. Pkd1 −/− with Pkd1 Mini transfer exhibits fewer cysts in proximal tubular segment but readily detectable glomerular cysts (Glom cyst) as observed in Pkd1 −/− kidneys. (I) Global quantification of cystic tubular segments relative to all tubular segments in Pkd1 −/− with Pkd1 Mini transfer. Pkd1 −/− mice with Pkd1 Mini markedly postponed the appearance of proximal cysts until at least P10 and delayed cysts in distal tubules until P5 and was insufficient to improve the percentage of collecting tubular cysts at P5. Newborn Pkd1 −/− shared with Figure 2 I. ♦♦♦♦ p < 0.0001, ♦♦♦ p < 0.001.

Article Snippet: Pkd1 levels were assessed with the TaqMan master mix and predesigned duplex gene expression assays for Pkd1 , Mm00465434_m1 (FAM-MGB) (cat. no. 4331182), and for Hprt1 , Mm00446968_m1 (VIC-MGB) (cat. no. 4448489) (Applied Biosystems), and Pkd2 levels with PowerUp SYBR Green using the following primers: forward, 5′-CGAGTCAGAAACGGATCCTGCTCCA, and reverse, 5′-CCAGTGACTGCTCCCATTCAGCTC.

Techniques: Expressing, Western Blot, Quantitative Proteomics, Comparison, Control, Immunofluorescence

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modeling Pkd1 gene-targeted strategies for correction of polycystic kidney disease

doi: 10.1016/j.omtm.2023.03.016

Figure Lengend Snippet: Endogenous Pkd1 tubular cell expression pattern in wild-type mouse kidney (A) Pkd1 cellular expression in wild-type kidney at P5 in individual tubular segments was carried out by in situ Pkd1 expression pattern detection (white dots; RNAscope) with co-detection by immunofluorescence of specific tubular markers. Wild-type proximal tubules correspond to very weak dot signals, distal and collecting tubules display substantially more abundant Pkd1 dot signals. (B) Wild-type kidney section at P20 with co-detection of Pkd1 by RNAscope and tubular segment markers. The top illustrates specifically Pkd1 signals from RNAscope ISH, tubular markers, and co-detection in kidneys. Proximal, distal, and collecting tubules with magnified region marked by asterisks with co-detection of Pkd1 dot signals are illustrated below. (C) Pkd1 expression in wild-type glomeruli at P5 and P20 by RNAscope. Distinct but weak Pkd1 signals are detected over mesangial cells at P5, which are of higher intensity at P20 and visible in visceral cells. Pkd1 expression in Pkd1 −/− glomeruli with Pkd1 wt2 transfer at P5 has a similar pattern with considerably more intense dot signals than wild-type glomeruli. Scale as indicated.

Article Snippet: Pkd1 levels were assessed with the TaqMan master mix and predesigned duplex gene expression assays for Pkd1 , Mm00465434_m1 (FAM-MGB) (cat. no. 4331182), and for Hprt1 , Mm00446968_m1 (VIC-MGB) (cat. no. 4448489) (Applied Biosystems), and Pkd2 levels with PowerUp SYBR Green using the following primers: forward, 5′-CGAGTCAGAAACGGATCCTGCTCCA, and reverse, 5′-CCAGTGACTGCTCCCATTCAGCTC.

Techniques: Expressing, In Situ, RNAscope, Immunofluorescence

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modeling Pkd1 gene-targeted strategies for correction of polycystic kidney disease

doi: 10.1016/j.omtm.2023.03.016

Figure Lengend Snippet: Spatial and cellular Pkd1 expression from transgene transfer in Pkd1 −/− kidneys alone or merged with individual or all tubular markers (A) Kidneys of Pkd1 −/− with one-copy SB Pkd1 transfer at P5 by RNAscope exhibit generally weak Pkd1 signals in non-cystic and cystic tubules, with occasional intense dot signals distinctive from wild-type kidneys. Scale: 50 μm. (B) Kidneys of Pkd1 −/− with high-copy SB Pkd1 transfer at P20 have strong Pkd1 dot signals in comparison with one-copy SB Pkd1 . Pkd1 signals with high-copy SB Pkd1 transfer are more intense in the majority of distal and collecting tubules than in proximal tubules. Signals are detected in non-cystic and small/intermediate cystic tubules and of mostly lower Pkd1 intensity in large cystic tubules. Scale: 50 μm. (C) Kidneys of Pkd1 −/− with Pkd1 Mini transfer at P5 display more diffuse and widespread Pkd1 signals with numerous dots of low intensity, whether of cystic or non-cystic tubules and regardless of the tubular segments. Large cysts in collecting tubules generally exhibit similar uniform low Pkd1 expression. Scale: 50 μm.

Article Snippet: Pkd1 levels were assessed with the TaqMan master mix and predesigned duplex gene expression assays for Pkd1 , Mm00465434_m1 (FAM-MGB) (cat. no. 4331182), and for Hprt1 , Mm00446968_m1 (VIC-MGB) (cat. no. 4448489) (Applied Biosystems), and Pkd2 levels with PowerUp SYBR Green using the following primers: forward, 5′-CGAGTCAGAAACGGATCCTGCTCCA, and reverse, 5′-CCAGTGACTGCTCCCATTCAGCTC.

Techniques: Expressing, RNAscope, Comparison

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modeling Pkd1 gene-targeted strategies for correction of polycystic kidney disease

doi: 10.1016/j.omtm.2023.03.016

Figure Lengend Snippet: Comparison of Pkd1 −/− gene-targeted transfer potential on PKD time-course progression or correction (A) Timeline of renal cyst initiation in Pkd1 −/− mice at e15.5 and death by birth within 5 days. Pkd1 −/− mice with Pkd1 Mini transfer display cysts at birth until P15, whereas those with one-copy SB Pkd1 transfer develop cysts later at P3 until P15. Pkd1 −/− mice with high-copy SB Pkd1 transfer initiate cysts between P10 and P15 and progress to severe cysts by ∼4 months. Pkd1 −/− mice with Pkd1 wt transfer have a completely rescued phenotype. (B) Schematic representation of cyst appearance and progression in different tubular segments, and severity over time for the three different transgene transfers. (C) Survival expectancy of Pkd1 −/− mice, estimated as 5 days, is markedly extended from e15.5 to P15 or equivalent to a 20 day period for both transfers, Pkd1 Mini or one-copy SB Pkd1 in Pkd1 −/− mice, an appraisal of significantly high impact. In Pkd1 −/− mice with high-copy SB Pkd1 transfer, the very slow kinetics of disease progression extended life expectancy by an impressive ∼25-fold. Pkd1 −/− mice with Pkd1 wt transfer are predicted to have a normal lifespan.

Article Snippet: Pkd1 levels were assessed with the TaqMan master mix and predesigned duplex gene expression assays for Pkd1 , Mm00465434_m1 (FAM-MGB) (cat. no. 4331182), and for Hprt1 , Mm00446968_m1 (VIC-MGB) (cat. no. 4448489) (Applied Biosystems), and Pkd2 levels with PowerUp SYBR Green using the following primers: forward, 5′-CGAGTCAGAAACGGATCCTGCTCCA, and reverse, 5′-CCAGTGACTGCTCCCATTCAGCTC.

Techniques: Comparison, Biomarker Discovery

Journal: iScience

Article Title: Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

doi: 10.1016/j.isci.2022.104359

Figure Lengend Snippet: Deletion of PC1 results in a switch from tubulogenesis to cyst formation and is accompanied by an increase of cAMP and a decrease in cell motility (A and B) PC1-competent clonal cells (PKD1 +/+ ) mainly formed tubule-like structures within a collagen matrix when incubated with control medium (Ctrl). In contrast, PC1-deficient cells (PKD1 −/− ; shown for two different clones #1 and #2) primarily formed cysts within the collagen matrix in the presence of control medium (Ctrl). Application of forskolin (FSK; 10μM) to PKD1 +/+ cells turned them into cyst-forming cells resembling the phenotype obtained by PC1-deletion under control condition. Incubation of PKD1 −/− cells with forskolin (FSK; 10μM) had no impact on their morphology. (A) provides the analysis of the ratio of cystic to tubule-like formed structures (analysis of 128 images from n = 4 individual experiments). (B) shows representative images after five days of culture within a collagen I matrix. (C) Measurements of cAMP concentrations revealed similar values in clonal PC1-competent cells (PKD1 +/+ ) compared to wild type cells (wt). PC1-deletion resulted in significantly elevated cAMP levels in PC1-deficient cells (PKD1 −/− #1 and #2) with PKD1 +/+ set as 100% (n = 6 individual experiments). Incubation of the cells with forskolin (FSK; 10μM) resulted in a significant increase of cAMP in PC1-competent and PC1-deficient cells to a similar level (n = 3 individual experiments). (D and E) Movement of spheroids of PC1-competent and PC1-deficent cells (shown for PKD1 −/− #1) within the collagen matrix was captured by live imaging of 30 spheroids per condition and cell type from n = 3 individual experiments for 48 h. Deletion of PC1 resulted in decreased motility. Incubation with forskolin (FSK; 10μM) also led to a reduction of motility in PC1-competent cells to a similar degree obtained from PC1-defcient cell under control condition. Application of forskolin to PC1-deficient spheroids had no additional effect. (D) Analysis of movements of spheroids within the matrix. (E) Representative traces of single spheroids within 48 h of imaging. ∗significant compared to PKD1 +/+ -Ctrl; § significant compared to PKD1 −/− #1-FSK and PKD1 −/− #2-FSK, respectively. Data are represented as mean ± SEM. See also Figures S1–S , and .

Article Snippet: Guide RNAs targeting Exon 2 of the Pkd1 gene were ligated with a pSpCas9(BB)-2A-puro-vector (PX459) V2.0 from Addgene (ID 62988; Watertown, MA, USA).

Techniques: Incubation, Control, Clone Assay, Imaging

Journal: iScience

Article Title: Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

doi: 10.1016/j.isci.2022.104359

Figure Lengend Snippet: cAMP leads to impaired capacity for tubule formation but is more pronounced in PC1-deficient cells PC1-competent (PKD1 +/+ ) and PC1-deficient cells (PKD1 −/− ) were cultured within a collagen matrix in the presence of forskolin (FSK; 10μM) where they formed cysts within four days. Thereafter PC1-competent cysts (n = 47 from three individual experiments) and PC1-deficient cysts (n = 75 from four individual experiments) were punctured with a pulled glass capillary microneedle and the use of a micromanipulator. Punctured cysts were imaged for 70 h as depicted in (A). (B) Analysis of the ratio of cysts forming tubules after injury. (C) Analysis of the length of the formed tubules within 70 h. (D) Outgrowth speed of the formed tubules by tracking the distance within 1000 min (measured from minute 100 to 1100 after injury). (E) Representative images showing tubular outgrowth in PC1-competent and PC1-deficient cysts. ∗significant compared PKD1 +/+ . Data are represented as mean ± SEM. See also Figure S9 , and .

Article Snippet: Guide RNAs targeting Exon 2 of the Pkd1 gene were ligated with a pSpCas9(BB)-2A-puro-vector (PX459) V2.0 from Addgene (ID 62988; Watertown, MA, USA).

Techniques: Cell Culture

Journal: iScience

Article Title: Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

doi: 10.1016/j.isci.2022.104359

Figure Lengend Snippet: Lack of PC1 along with increase of cAMP leads to defective cell migration (A) Tracks of individual cells (50 cells per condition from n = 3 individual experiments) imaged for 6 h in wound healing assays starting 2 h after wounding the cell monolayer. Deletion of PC1 (PKD1 −/− ; red) resulted in reduced migration. The same was obtained when incubating PC1-competent cells (PKD1 +/+ ; blue) with forskolin. (B) Representative images of wound healing assays showing the invaded area of cells within 6 h. (C) Quantification of the euclidean distance of the individually tracked cells (left) and the ratio of euclidean distance in relation to the accumulated distance (right) indicating significant impairment of directed cell migration upon loss of PC1 or application of forskolin in PC1-competent cells. ∗significant compared to PKD1 +/+ -Ctrl. Data are represented as mean ± SEM. See also Figure S6 , and .

Article Snippet: Guide RNAs targeting Exon 2 of the Pkd1 gene were ligated with a pSpCas9(BB)-2A-puro-vector (PX459) V2.0 from Addgene (ID 62988; Watertown, MA, USA).

Techniques: Migration

Journal: iScience

Article Title: Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

doi: 10.1016/j.isci.2022.104359

Figure Lengend Snippet: Loss of PC1 and increase of cAMP lead to dense cell aggregation and reduction of disseminating single cells PC1-competent cells (PKD1 +/+ ) and PC1-deleted cells (PKD1 −/− ) were cultured at low density (3200 cells/cm 2 ) and analyzed with regard to their growing pattern. (A) PC1-competent cells kept growing in a disseminated pattern forming small colonies with a significant amount of single cluster-independent cells. PC1-deleted cells resulted in dense and evenly bordered cell aggregations with only a few single cells being detectable outside these cell formations. Forskolin (FSK; 10μM) significantly reduced the number of single PC1-competent and PC1-deficient cells (analysis of 72 images from n = 3 individual experiments). (B) Voronoi-based analysis of cell distance between clustering cells (defined as > two cells). Cell distance was significantly higher in PC1-competent cells compared to PC1-deficient cells and PC1-competent cells in the presence of forskolin. (C) Analysis of cell density within bigger cell aggregates (comprising 10 - 50 cells/cluster). Cells were significantly less dense within the clusters of PC1-competent cells under control condition compared to PC1-deficient cells or PC1-competent cells in the presence of forskolin. (D) Representative images after 48 h of culture of PC1-competent cells (PKD1 +/+ ) and PC1-deficient cells (PKD1 −/− ) under control condition (Ctrl) or in the presence of forskolin (FSK; 10μM). Row 1 shows representative images of DAPI-stained cells. In each image, one cell cluster was marked by a yellow outline in order to facilitate finding of the corresponding parts in the Voroni-based animations and heat map analyses. Row 2 shows Voronoi-based animation of cell clusters (defined as > two cells; white) and single cells (red). Row 3 shows heatmap analysis of cells visualizing cell density defined as blue = low cell density and red = high cell density. ∗significant compared to PKD1 +/+ -Ctrl. # significant compared to PKD1 −/− -Ctrl. Data are represented as mean ± SEM. See also Figures S7 and .

Article Snippet: Guide RNAs targeting Exon 2 of the Pkd1 gene were ligated with a pSpCas9(BB)-2A-puro-vector (PX459) V2.0 from Addgene (ID 62988; Watertown, MA, USA).

Techniques: Cell Culture, Control, Staining

Journal: iScience

Article Title: Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

doi: 10.1016/j.isci.2022.104359

Figure Lengend Snippet: PC1 deletion and cAMP increase lead to impaired epithelial tubular cell orientation in metanephric mouse kidneys Metanephric kidneys (n = 3 per condition) from KspCreER T2 : Pkd1 lox;lox mice were dissected at embryonic day 13.5 and either incubated with control medium in order to preserve PC1 expression ( Pkd1 +/+ ) or medium supplemented with hydroxytamoxifen (500 nM) resulting in tubule-specific deletion of PC1 ( Pkd1 −/− ). Thereafter, kidneys were cultured ex vivo for five days either incubated with control medium (Ctrl) or in the presence of forskolin (FSK; 10μM). (A) Violin plots illustrating the deviation of the tubule cell’s longitudinal axis from the longitudinal tubular axis with a reference angle set = 90° (n = 30 tubules per condition with a mean of 10 analyzed cells per tubule). (B and C) Representative images of metanephric kidneys stained for E-Cadherin illustrating tubule epithelial cell borders and binarized images with depicted longitudinal cell axes. ∗significant compared to PKD1 +/+ -Ctrl. Data are represented as mean ± SEM.

Article Snippet: Guide RNAs targeting Exon 2 of the Pkd1 gene were ligated with a pSpCas9(BB)-2A-puro-vector (PX459) V2.0 from Addgene (ID 62988; Watertown, MA, USA).

Techniques: Incubation, Control, Expressing, Cell Culture, Ex Vivo, Staining

Journal: iScience

Article Title: Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

doi: 10.1016/j.isci.2022.104359

Figure Lengend Snippet:

Article Snippet: Guide RNAs targeting Exon 2 of the Pkd1 gene were ligated with a pSpCas9(BB)-2A-puro-vector (PX459) V2.0 from Addgene (ID 62988; Watertown, MA, USA).

Techniques: Recombinant, Luciferase, Plasmid Preparation, Cloning, Software, Chemotaxis Assay, Migration, Sequencing, Cell Culture