Journal: Stem cell research

Article Title: Induced pluripotent stem cell line from a mouse model of human azoospermia with a frameshift mutation Tex11 _1260Ins(TT)

doi: 10.1016/j.scr.2022.102728

Figure Lengend Snippet: Characterization of the Tex11_1260Ins(TT) induced pluripotent stem cell (iPSC) line derived from an infertile male mouse model. (A) Phase contrast image showing normal morphology of Tex11_1260Ins(TT) iPSCs growing on a feeder-coated plate (scale bar = 50 μm). (B) Genotyping PCR products of Tex11_1260Ins(TT) iPSCs to confirm their origin from Tex11_1260Ins(TT) mouse fibroblasts. Wild-type control iPSCs (labeled as (+) WT) were used as a control. (C) Sanger sequencing of PCR products for wild type iPSCs, Tex11_1260Ins(TT) fibroblasts , and Tex11_1260Ins(TT) iPSCs to confirm their genotype as wild-type (GATG) or mutant (TTGGTA). (D) PCR results confirming the sex of Tex11_1260Ins(TT) iPSCs as male using F Sry -R Sry primer pair specific for the Sry gene. Cells of male (M) origin or female (F) origin were used as controls. Fc-Rv primer pair was used to confirm presence of DNA in all cell samples. (E) RT-PCR result showing absence of sendai virus (SeV) in Tex11_1260Ins(TT) iPSCs at passage 16. Tex11_1260Ins(TT) iPSCs at passage 3 were used as positive control for SeV. GAPDH housekeeping gene was used as a control for RT-PCR analysis. (F) Tex11_1260Ins(TT) iPSCs showing normal karyotype of 40 chromosomes (mouse). (G) Immunofluorescent staining of Tex11_1260Ins(TT) iPSCs showing positive staining for pluripotent markers Alkaline phosphatase (AP), SSEA1, SOX2, OCT4, NANOG, DPPA2. (H) Flow cytometry analysis showing no difference in percentage of SSEA1+, SOX2+, or OCT4 + Tex11_1260Ins(TT) iPSCs compared to control iPSCs. (I) Teratoma assay analysis confirming tri-lineage (endoderm, mesoderm, ectoderm) differentiation potential of transplanted Tex11_1260Ins(TT) iPSCs.

Article Snippet: A published mouse iPSC line (ALSTEM) was used as the control.

Techniques: Derivative Assay, Control, Labeling, Sequencing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Virus, Positive Control, Staining, Flow Cytometry

Journal: Stem cell research

Article Title: Induced pluripotent stem cell line from a mouse model of human azoospermia with a frameshift mutation Tex11 _1260Ins(TT)

doi: 10.1016/j.scr.2022.102728

Figure Lengend Snippet: Characterization and validation.

Article Snippet: A published mouse iPSC line (ALSTEM) was used as the control.

Techniques: Biomarker Discovery, Immunocytochemistry, Staining, Flow Cytometry, Mutagenesis, Sequencing, Amplification, Positive Control, Negative Control, Derivative Assay, Plasmid Preparation, Modification, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Toward Xeno-Free Differentiation of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells

doi: 10.3390/ijms23031312

Figure Lengend Snippet: Differentiation toward small intestinal epithelium ( a ) Intracellular localization of small intestinal epithelial cell markers in SIEC-iPSCs cultured either on Geltrex or laminin 511. Caco-2 cells grown on Geltrex served as a control. Enterocyte marker proteins PEPT1 (green) and villin (green), and enteroendocrine cell marker chromogranin A (CGA, green) were visualized with indirect immunofluorescence and confocal microscopy. In samples stained with Chromogranin A, the arrowhead in Geltrex, and arrow in laminin iPSC-SIEC samples point to extracellular matrix. Number of biological replicates n = 4, number of technical replicates n = 2. Scalebar 20 µm. ( b ) The expression of enterocyte marker genes PEPT1, villin and CYP3A4 was analyzed with qRT-PCR. The expression of iPSC-SIECs were calibrated against iPSC-SIEC cultured on Geltrex. Number of biological replicates n = 3, number of technical replicates in every biological replicate n = 3. Data expressed as mean ± SD. The statistical significance: *** ( p ≤ 0.001).

Article Snippet: During the time of the experiment, the karyotypes of iPSC line was normal (Fimlab Laboratories, Tampere, Finland). iPSCs were maintained on Geltrex (Gibco; Thermo Fisher Scientific, Waltham, MA, USA; diluted 1:100) coated Cell-Bind treated 6-well plates (Corning, Corning, NY, USA) in mTeSR1 medium (StemCell Technologies, Vancouver, BC, Canada) with penicillin–streptomycin (P/S; Gibco, Paisley, UK).

Techniques: Cell Culture, Control, Marker, Immunofluorescence, Confocal Microscopy, Staining, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Toward Xeno-Free Differentiation of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells

doi: 10.3390/ijms23031312

Figure Lengend Snippet: Small intestinal epithelial cell specific functionality assessed with dipeptide uptake assay. Posterior definitive endoderm differentiated iPSCs were cultured on Geltrex or Laminin511 and differentiated toward small intestinal epithelial cells. Caco-2 cells cultured on Geltrex served as control. ( a ) The intensity of fluorescence analyzed from micrographs after dipeptide (D-Ala-Leu-Lys-AMCA) uptake analyses +/− ibuprofen treatment. ( b ) iPSC-SIEC cultured on Geltrex, CTRL i.e., without ibuprofen, ( c ) iPSC-SIEC cultured on laminin, CTRL, i.e., without ibuprofen, ( d ) Caco-2 cultured on Geltrex, CTRL, i.e., without ibuprofen, ( e ) iPSC-SIEC cultured on Geltrex, with ibuprofen, ( f ) iPSC-SIEC cultured on laminin, with ibuprofen, ( g ) Caco-2 cultured on Geltrex, with ibuprofen. Number of biological replicates n = 3–4, number of technical replicates n = 3. Data expressed as mean ± SD. The statistical significance: ** ( p ≤ 0.005) and *** ( p ≤ 0.001) indicating the significance between the indicated samples.

Article Snippet: During the time of the experiment, the karyotypes of iPSC line was normal (Fimlab Laboratories, Tampere, Finland). iPSCs were maintained on Geltrex (Gibco; Thermo Fisher Scientific, Waltham, MA, USA; diluted 1:100) coated Cell-Bind treated 6-well plates (Corning, Corning, NY, USA) in mTeSR1 medium (StemCell Technologies, Vancouver, BC, Canada) with penicillin–streptomycin (P/S; Gibco, Paisley, UK).

Techniques: Cell Culture, Control, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Toward Xeno-Free Differentiation of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells

doi: 10.3390/ijms23031312

Figure Lengend Snippet: The functionality of efflux transport proteins. The functionality of efflux transporters was assessed with calcein retention assay in the absence (=CTRL) or presence of efflux protein inhibitors Cyclosporin A or verapamil from the small intestinal epithelial cells differentiated from posterior definitive endoderm on Geltrex or laminin511. ( a ) Functionality, which is seen as retention, is expressed as a percentage of fluorescence relative to the control (control = 100%, is marked with the red line. ( b – j ) micrographs taken to quantitate retention efficiency ( b ) iPSC-SIEC cultured on Geltrex, without inhibitors (CTRL), ( c ) iPSC-SIEC cultured on laminin, without inhibitors (CTRL), ( d ) Caco-2 cultured on Geltrex, without inhibitors (CTRL), ( e ) iPSC-SIEC cultured on Geltrex, with Cyclosporin A, ( f ) iPSC-SIEC cultured on laminin, with Cyclosporin A, ( g ) Caco-2 cultured on Geltrex, with Cyclosporin A. ( h ) iPSC-SIEC cultured on Geltrex, with Verapamil, ( i ) iPSC-SIEC cultured on laminin, with Verapamil, ( j ) Caco-2 cultured on Geltrex, with Verapamil. Scalebar 100 µm. Number of biological replicates n = 3–4, number of technical replicates n = 3. Data expressed as mean ± SD. The statistical significance: * ( p ≤ 0.05), ** ( p ≤ 0.005) and *** ( p ≤ 0.001) indicating the significance between the indicated samples.

Article Snippet: During the time of the experiment, the karyotypes of iPSC line was normal (Fimlab Laboratories, Tampere, Finland). iPSCs were maintained on Geltrex (Gibco; Thermo Fisher Scientific, Waltham, MA, USA; diluted 1:100) coated Cell-Bind treated 6-well plates (Corning, Corning, NY, USA) in mTeSR1 medium (StemCell Technologies, Vancouver, BC, Canada) with penicillin–streptomycin (P/S; Gibco, Paisley, UK).

Techniques: Fluorescence, Control, Cell Culture

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) The mRNAs levels of endogenous SOX2, OCT3/4, LIN28 and NANOG in the iPSC lines were quantified and normalized relative to hESC line H9. ( B ) The amount of transcripts of the episomal reprograming vectors were quantified for the iPSC lines between passage number 12 and number 15, using human skin fibroblasts nucleofected with the same vectors as a positive control. DOI: http://dx.doi.org/10.7554/eLife.13073.005

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Positive Control

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) NKX2.1 protein expression was examined by immunocytochemistry in ventral telencephalic neural rosettes derived from a number of control and Dravet iPSC lines. Scale bars, 200 microns. ( B ) OLIG2 protein expression overlaps with NKX2.1 in ventral telencephalic progenitors derived from the hESC line H9. Scale bar, 100 microns. ( C ) Comparing NKX2.1, COUP-TFII and SP8 mRNA expression between dorsal and ventral telencephalic neural rosettes derived from control hESC line H9, Dravet iPSC line 6358–2, and Control iPSC line 8402–2 WT9. Data were quantified by the delta-delta Ct method, using GAPDH as the reference gene and undifferentiated H9 ESCs as the baseline control. It is known that SP8 is weakly expressed in progenitors of excitatory neurons in the developing dorsal telencephalon , which likely corresponds to the detection of SP8 in the dorsal rosette samples in this experiment. The samples represent two neural differentiation experiments (one for H9 and 6358–2 and one for 8402–2) that were independent of those described in . DOI: http://dx.doi.org/10.7554/eLife.13073.014

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Expressing, Immunocytochemistry, Derivative Assay, Control

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) An example for the immunostaining of the inhibitory neurotransmitter GABA (in green) and general neuronal marker MAP2 (in red) in differentiated inhibitory neurons (top) and excitatory neurons (bottom). The cell line in the images is hESC-H9. Scale bars, 50 microns. ( B ) Representative images showing a Calretinin + GAD67 + inhibitory neuron and a Somatostatin + GABA + inhibitory neuron. The cell line in the images is hESC-H9. ( C ) Percentage of Calretinin + or Somatostatin + inhibitory neurons from differentiation of hESC-H9 and control iPSC line 8858-C. ( D ) In inhibitory neuron cultures the expression of lentiviral reporter Dlxi1/2b-GFP overlapped with GABA immunostaining; on the contrary, in excitatory neuron cultures, CaMKII-GFP labeling and GABA immunostaining were mutually exclusive. Scale bars, 20 microns. ( E ) Around 65% of Dlxi1/2b-GFP + neurons were stained positive for GABA. Data represent two control iPSC lines 8858-C and 6593–7. ( F ) By single-cell RT-PCR paired with patch clamp recording, around 70% of Dlxi1/2b-GFP neurons express either GAD65 or GAD67, markers of inhibitory neurons. Data represent all Control (upper pie chart) and Dravet Syndrome (lower pie chart) pluripotent stem cell lines examined in this study. ( G ) Representative traces showing time-dependent maturation of the action potential firing pattern in inhibitory neurons. The cell line in this example is control iPSC 8858–3. ( H ) An example for the isolation of Gabazine-sensitive, spontaneous inhibitory synaptic currents (sIPSCs, insets in blue) and NBQX-sensitive, spontaneous excitatory synaptic currents (sEPSCs, insets in red) in cultures of differentiated inhibitory neurons. The detection of sEPSCs reflects the presence of a small fraction of excitatory neurons which likely arise from the low-percentage of PAX6 + progenitors in the ventral telencephalon patterning protocol . The cell line in this example is Dravet iPSC 6358–3. ( I ) Visualization of a hESC-H9-derived inhibitory neuron engrafted to the CA3 region of a rat hippocampal slice and co-cultured with the slice for 2 months. The human neuron was labeled with the lentiviral vector pLenti-Syn1-ChR2(H134R)-YFP. Rat neurons were visible by Ankyrin-G immunostaining (in red). Scale bar, 25 microns. ( J ) Under 470-nm light pulses, Gabazine-sensitive inhibitory postsynaptic currents (IPSCs, top trace) were detected in a rat neuron on a hippocampal slice engrafted with ChR2-YFP-expressing, hESC-H9-derived inhibitory neurons. Each trace represents the average of 20 consecutive trials (light pulses). DOI: http://dx.doi.org/10.7554/eLife.13073.015

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Immunostaining, Marker, Control, Expressing, Labeling, Staining, Reverse Transcription Polymerase Chain Reaction, Patch Clamp, Isolation, Derivative Assay, Cell Culture, Plasmid Preparation

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) Inhibitory neuron cultures derived from control iPSC line 8402–2 WT9 were fixed before plating onto rat astrocytes and stained for NKX2.1 (in green, using Novacastra antibody TTF‐1‐L‐CE), Calretinin (in red, using Swant antibody 7697) and nuclei (Hoechst, in blue). The arrows point to cells with neuronal morphology and co-expressing NKX2.1 and Calretinin. Scale bars, 50 microns. ( B ) The cells described in ( A ) were further differentiated in the presence of rat astrocytes and stained for GAD67 (in white, using EMD Millipore antibody AB5406) and SP8 (in red, using Sigma antibody HPA054006). In a total of 199 GAD67 + , neuronal-looking cells that we analyzed, 81% (161 out of 199) was negative for SP8 staining, and the remaining 19% (38 out of 199) showed a very weak signal (one example shown in ( B ), highlighted by open arrow head). Scale bar, 100 microns. ( C ) As a positive control for SP8 detection, we examined primary cultures of rat cortical neurons (three weeks in vitro) in the same experiment under identical staining and imaging conditions. Robust SP8 staining was seen in a subpopulation of GAD67 + inhibitory neurons (arrow head). Scale bar, 100 microns. Because the mutually exclusive expression of SP8 and NKX2.1 distinguishes between the CGE- and MGE-origin of inhibitory interneurons, this set of supplementary data further support that the human iPSC-derived inhibitory neurons in this study mostly resemble a MGE origin, and that Calretinin expression may arise in MGE-lineage inhibitory neurons within the time scale of the study. DOI: http://dx.doi.org/10.7554/eLife.13073.016

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Derivative Assay, Control, Staining, Expressing, Positive Control, In Vitro, Imaging

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) An example of I Na measured in an inhibitory neuron derived from a control iPSC line. The membrane depolarization steps were set between -92 mV and a range of values starting from -72 mV and ending at +38 mV, with 5 mV/step increments. ( B ) Complete blockade of I Na by 0.8 µM TTX. ( C and D ) I Na was indistinguishable between control and Dravet excitatory neurons. The example I Na traces in ( C ) were selected to represent the median values of all control excitatory neurons and all Dravet excitatory neurons, respectively. The current trace from the Dravet neuron was scaled relative to the control neuron according to whole-cell capacitance values. Panel ( D ) describes the current-voltage (I-V) relationships in control (n = 45 cells, 3 subjects) and Dravet (n = 45 cells, 2 subjects) excitatory neurons. p = 0.8225 by t-test of the maximal I Na amplitude. ( E and F ) I Na amplitude was reduced in Dravet inhibitory neurons relative to control inhibitory neurons. The example I Na traces in ( E ) were selected the same way as in ( C ). Panel ( F ) describes the I-V relationships of control (n = 28 cells, 4 subjects) and Dravet (n = 20 cells, 2 subjects) inhibitory neurons. p = 0.034 by t-test for the maximal I Na amplitude. All error bars are standard errors of the mean. See for detailed statistical analysis. DOI: http://dx.doi.org/10.7554/eLife.13073.020 10.7554/eLife.13073.021 Figure 4—source data 1. I Na values quantified in . DOI: http://dx.doi.org/10.7554/eLife.13073.021

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Derivative Assay, Control, Membrane

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: The pool of inhibitory neurons (n = 60) in this analysis were derived from hESC-H9 and control iPSC lines and co-cultured with rat astrocytes until around Day 90. Illustrated here are current clamp traces at the threshold current injection (i.e. the rheobase, in red) and at a supra-threshold current that is two or three times of the threshold current (in black). We also plotted the response to a−10 pA hyperpolarizing current (downward trace in black). All current injections are 1000-msec long. By their supra-threshold firing patterns, the inhibitory neurons are classified as accommodating, non-accomodating, stuttering, immature, and single-AP. Panel ( A ) is a pie chart summary of the percentage representation of the five patterns. Panels ( B ) through ( F ) are representative neurons for each pattern. DOI: http://dx.doi.org/10.7554/eLife.13073.018

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Derivative Assay, Control, Cell Culture, Injection

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) Differential expression of Na v 1.1 mRNA between cultures of telencephalic excitatory neurons and inhibitory neurons. Quantification of Na v 1.1 mRNA was based on real-time PCR and normalization to the auxiliary Na channel subunit NaVβ3. For control iPSC line 8858–3, n = 6 culture wells of excitatory neurons, n = 5 culture wells of inhibitory neurons. For the other cell lines, one culture well of excitatory neurons and one culture well of inhibitory neurons were assessed pairwise. ( B ) Visually identifying Dlxi1/2b-GFP neurons that co-express Na v 1.1-shRNAs with a tRFP tracer. Scale bar, 10 microns. Similar tracing was performed for CaMKII-GFP-labeled excitatory neurons co-expressing hNa v 1.1-shRNAs. ( C ) Visually identifying Dlxi1/2b-GFP neurons that co-express a doxycycline-inducible Na v 1.1 cDNA with a mCherry tracer. Scale bar, 25 microns. ( D ) Na v 1.1- shRNA1 and Na v 1.1-shRNA2 reduced Na + currents (I Na ) in hESC-H9 derived Dlxi1/2b-GFP + neurons, but not in CaMKII-GFP + neurons. Conversely, the Na v 1.1 cDNA restored the I Na in Dravet Dlxi1/2b-GFP + neurons (Cell line: 6358–2). F(7, 57) = 5.636, p<0.0001 by ANOVA for the entire dataset. Individual p values from post hoc Sidak’s multiple comparisons are indicated on the graph. ( E ) Na v 1.1-shRNAs suppressed action potential firing capacity in hESC-H9 derived Dlxi1/2b-GFP + neurons. F (1, 270) = 13.24, p = 0.0003 by two-way ANOVA for the effect of Na v 1.1-shRNAs on spike number over the range of current injections. ( F ) The Na v 1.1 cDNA restored action potential firing capacity in Dlxi1/2b-GFP + neurons derived from Dravet iPSC line 6358–2. F (1, 135) = 26.69, p<0.0001 by two-way ANOVA for the contribution of Na v 1.1 cDNA to spike number over the range of current injections. All error bars are standard errors of the mean. DOI: http://dx.doi.org/10.7554/eLife.13073.031 10.7554/eLife.13073.032 Figure 6—source data 1. I Na values quantified in , and action potential numbers quantified in . DOI: http://dx.doi.org/10.7554/eLife.13073.032

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Quantitative Proteomics, Real-time Polymerase Chain Reaction, Control, Labeling, Expressing, Derivative Assay

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) Immunocytochemistry for pluripotency markers in control iPSC lines 8858-C and 8858–3. OCT3/4 and NANOG are localized to the cell nucleus; TRA-1-60 and SSEA-3 are localized to the cell surface; TRA-2-49 is localized in the cytoplasm. Scale bars, 200 microns. ( B ) Immunocytochemistry for pluripotency markers in control iPSC line 8402–2 WT9. Upper row, co-expression of pluripotent stem cell markers OCT3/4 (in green) and TRA-1-60 (in red). Lower row, co-expression of pluripotent stem cell markers NANOG (in green) and SSEA-4 (in red, cell surface localization). Scale bars, 200 microns. ( C ) Summary of characterizations of all control iPSC lines with regard to the silencing of reprogramming vectors and genome integrity. DOI: http://dx.doi.org/10.7554/eLife.13073.007

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Immunocytochemistry, Control, Expressing

Journal: Genes

Article Title: NEK1 Facilitates Cohesin Removal during Mammalian Spermatogenesis

doi: 10.3390/genes2010260

Figure Lengend Snippet: Nek1 kat2J/kat2J mice show decreased testes size. ( A ) Photograph of both Nek1 kat2J/kat2J and wild type littermate testis; ( B ) Wild type (black) and mutant Nek1 kat2J/kat2J (gray) testes and heart sizes, shown as a percentage of total body weights.

Article Snippet: NEK1 is highly expressed in murine germ cells, predominantly in the testes in spermatogonial cells and in prophase I stage spermatocytes [ , ] and at least two distinct mutant mouse lines for Nek1 , generated at the Jackson laboratories, display severely impaired fertility [ – ], indicating a probable involvement in meiotic progression.

Techniques: Mutagenesis

Journal: Genes

Article Title: NEK1 Facilitates Cohesin Removal during Mammalian Spermatogenesis

doi: 10.3390/genes2010260

Figure Lengend Snippet: 3 week old Nek1 kat2J/kat2J mice show disorganized testes morphology. 3 week old wild type ( A , C ) and Nek1 kat2J/kat2J testes ( B , D - F ) were stained with H&E ( A , B , E ) or TRA-98 antibody ( C , D , F ). Empty tubules are shown by the asterisks and empty tubules corresponding to those with no germ cell staining by the arrows.

Article Snippet: NEK1 is highly expressed in murine germ cells, predominantly in the testes in spermatogonial cells and in prophase I stage spermatocytes [ , ] and at least two distinct mutant mouse lines for Nek1 , generated at the Jackson laboratories, display severely impaired fertility [ – ], indicating a probable involvement in meiotic progression.

Techniques: Staining

Journal: Genes

Article Title: NEK1 Facilitates Cohesin Removal during Mammalian Spermatogenesis

doi: 10.3390/genes2010260

Figure Lengend Snippet: 8 week old Nek1 kat2J/kat2J mice show disorganized testes morphology and an increase in apoptotic cells. 8 week old wild type ( A , C , E ) and Nek1 kat2J/kat2J ( B , D , F ) testes were stained with H&E ( A , B ), TRA-98 ( C , D ) or TUNEL ( E , F ). The sperm tails in the wild type seminiferous tubules are shown with an asterisk, and the TUNEL positive cells in metaphase by the arrows and in the insets.

Article Snippet: NEK1 is highly expressed in murine germ cells, predominantly in the testes in spermatogonial cells and in prophase I stage spermatocytes [ , ] and at least two distinct mutant mouse lines for Nek1 , generated at the Jackson laboratories, display severely impaired fertility [ – ], indicating a probable involvement in meiotic progression.

Techniques: Staining, TUNEL Assay

Journal: Genes

Article Title: NEK1 Facilitates Cohesin Removal during Mammalian Spermatogenesis

doi: 10.3390/genes2010260

Figure Lengend Snippet: Nek1 kat2J/kat2J spermatocytes undergo normal synapsis. Wild type (not shown) and Nek1 kat2J/kat2J testes were stained with a variety of antibodies against meiotic prophase I proteins. Shown here is staining with SYCP3 (red), either SYCP1 or SYCP2 (green) and DAPI (blue) during the first four stages of prophase I; leptonema, zygonema, pachynema and diplonema.

Article Snippet: NEK1 is highly expressed in murine germ cells, predominantly in the testes in spermatogonial cells and in prophase I stage spermatocytes [ , ] and at least two distinct mutant mouse lines for Nek1 , generated at the Jackson laboratories, display severely impaired fertility [ – ], indicating a probable involvement in meiotic progression.

Techniques: Staining

Journal: Genes

Article Title: NEK1 Facilitates Cohesin Removal during Mammalian Spermatogenesis

doi: 10.3390/genes2010260

Figure Lengend Snippet: Nek1 kat2J/kat2J spermatocytes undergo normal double-strand break (DSB) initiation and repair. Wild type (not shown) and Nek1 kat2J/kat2J testes were stained with a variety of antibodies against meiotic prophase I proteins. Shown here is staining with SYCP3 (green), and either RAD51 or MHSH4 (red) during the zygotene and pachytene stages of prophase I.

Article Snippet: NEK1 is highly expressed in murine germ cells, predominantly in the testes in spermatogonial cells and in prophase I stage spermatocytes [ , ] and at least two distinct mutant mouse lines for Nek1 , generated at the Jackson laboratories, display severely impaired fertility [ – ], indicating a probable involvement in meiotic progression.

Techniques: Staining

Journal: Genes

Article Title: NEK1 Facilitates Cohesin Removal during Mammalian Spermatogenesis

doi: 10.3390/genes2010260

Figure Lengend Snippet: NEK1 protein acts later than FKBP6 in prophase I cells. ( A ) Wild type and Nek1 kat2J/kat2J testes sections were stained with an antibody to FKBP6 (brown stain) and counter stained with Hematoxylin; ( B ) Wild type and Nek1 kat2J/kat2J pachytene spermatocytes stained with anti-SYCP3 (red), anti-FKBP6 (green) and DAPI (blue).

Article Snippet: NEK1 is highly expressed in murine germ cells, predominantly in the testes in spermatogonial cells and in prophase I stage spermatocytes [ , ] and at least two distinct mutant mouse lines for Nek1 , generated at the Jackson laboratories, display severely impaired fertility [ – ], indicating a probable involvement in meiotic progression.

Techniques: Staining

Journal: Genes

Article Title: NEK1 Facilitates Cohesin Removal during Mammalian Spermatogenesis

doi: 10.3390/genes2010260

Figure Lengend Snippet: Cohesin protein SMC3 is not removed in the proper temporal manner in Nek1 kat2J/kat2J spermatocytes. Wild type (A–D) and Nek1 kat2J/kat2J (E–H) spermatocytes were stained with anti-SYCP3 (green), anti-SMC3 (red) and CREST autoimmune serum (blue). Cells in leptonema (A, E), zygonema (B, F), pachynema (C, G) and diplonema (D, H) were assessed for SMC3 localization (gray panels show SMC3 staining alone). The persistent XY chromosome staining of SMC3 in wild type cells is shown by the arrow. Wild type (I) and Fkbp6 −/− (J) spermatocytes were also stained with anti-SYCP3 (green), anti-SMC3 (red) and CREST autoimmune serum (blue), however only the diplotene stage is shown here.

Article Snippet: NEK1 is highly expressed in murine germ cells, predominantly in the testes in spermatogonial cells and in prophase I stage spermatocytes [ , ] and at least two distinct mutant mouse lines for Nek1 , generated at the Jackson laboratories, display severely impaired fertility [ – ], indicating a probable involvement in meiotic progression.

Techniques: Staining

Journal: Genes

Article Title: NEK1 Facilitates Cohesin Removal during Mammalian Spermatogenesis

doi: 10.3390/genes2010260

Figure Lengend Snippet: REC8 and STAG3 cohesin localization in Nek1 kat2J/kat2J spermatocytes. Nek1 kat2J/kat2J prophase I spermatocytes were stained with anti-SYCP3 (green) and either anti-REC8 (A, B), or anti-STAG3 (C, D) (red or gray).

Article Snippet: NEK1 is highly expressed in murine germ cells, predominantly in the testes in spermatogonial cells and in prophase I stage spermatocytes [ , ] and at least two distinct mutant mouse lines for Nek1 , generated at the Jackson laboratories, display severely impaired fertility [ – ], indicating a probable involvement in meiotic progression.

Techniques: Staining

Journal: Genes

Article Title: NEK1 Facilitates Cohesin Removal during Mammalian Spermatogenesis

doi: 10.3390/genes2010260

Figure Lengend Snippet: Chromosomes fail to align on the metaphase spindle correctly in Nek1 kat2J/kat2J spermatocytes. Nek1 kat2J/kat2J and wild type testis sections were stained with H&E, and cells undergoing metaphase I imaged. Wild type cells generally show proper alignment of chromosomes on the spindle (left), whereas the majority of cells in Nek1 kat2J/kat2J testis look abnormally aligned or have lagging chromosomes (arrow).

Article Snippet: NEK1 is highly expressed in murine germ cells, predominantly in the testes in spermatogonial cells and in prophase I stage spermatocytes [ , ] and at least two distinct mutant mouse lines for Nek1 , generated at the Jackson laboratories, display severely impaired fertility [ – ], indicating a probable involvement in meiotic progression.

Techniques: Staining