fad complete medium  (PAN - Biotech)

Journal: Molecular Therapy

Article Title: Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing

doi: 10.1016/j.ymthe.2019.03.007

Figure Lengend Snippet: COL7A1 E80 Dual RNA Guide CRISPR/Cas9 Deletion Strategy (A) Scheme of the strategy. Single CRISPR RNA guides were designed to induce Cas9-mediated DNA double-strand breaks within selected COL7A1 intron sequences flanking exon 80 (U-Guides and D-Guides). NHEJ repair leads to intron-intron rejoining with concomitant E80 deletion and restoration of COL7A1 reading frame and truncated C7 expression. (B) CRISPR guide design. sgRNA (sgRNAs 1, 2, 3, and 4) sequences and alignment to E80-flanking sequences E80 are shown. The protospacer adjacent motif (PAM) is indicated in a darker color. (C) Predicted amplicon sizes for E80 deletions corresponding to each sgRNA pair combination. (D) PCR analysis of genomic DNA from RDEB keratinocytes from patient P1 treated with the RNP complexes containing the different sgRNA pair combinations. The sg2 + sg3 pair yielded the highest proportion of excised E80 according to the intensity of the lower 440-bp band. Deletion ratios assessed by densitometry are shown in each lane. “A” and “B” refer to the two different electroporation conditions tested.

Article Snippet: Primary human RDEB and healthy donor keratinocytes were cultured as previously described., Human primary RDEB keratinocytes from patients P1 and P2 were plated onto lethally irradiated 3T3-J2 cells and cultured in keratinocyte growth complete FAD (cFAD) medium (KCa), a 3:1 mix of DMEM and Ham’s F12 medium (Gibco-BRL, Barcelona, Spain) containing fetal bovine calf serum (Hyclone, GE Healthcare, Logan, UT) (10%), penicillin-streptomycin (1%), glutamine (2%), insulin (5 μg/mL; Sigma-Aldrich), adenine (0.18 mmol/L; Sigma-Aldrich), hydrocortisone (0.4 μg/mL; Sigma-Aldrich), cholera toxin (0.1 nmol/L; Sigma-Aldrich), triiodothyronine (2 nmol/L; Sigma-Aldrich), epidermal growth factor (EGF) (10 ng/mL; Sigma-Aldrich), and Y-27632 Rho-associated protein kinase (ROCK) inhibitor (Sigma-Aldrich) at 10 μM.

Techniques: CRISPR, Expressing, Amplification, Electroporation

Journal: Molecular Therapy

Article Title: Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing

doi: 10.1016/j.ymthe.2019.03.007

Figure Lengend Snippet: Collagen VII mRNA Expression of ΔE80 Gene-Edited RDEB Keratinocytes (A) RT-PCR analysis of COL7A1 transcripts amplified with primers in exons 78–84. Wild-type/c.6527insC unedited transcripts produced a 240/241-bp band (non-modified, n-m) found in all RNA samples. A smaller 205-bp band corresponding to transcripts lacking exon 80 was detected in samples from edited cells. M, DNA Molecular Weight Marker IX (Sigma-Aldrich) molecular weight marker; HK, healthy human keratinocytes; P1, patient keratinocytes; H 2 O, negative control without cDNA. (B) Representative sequence chromatograms showing the two different resulting transcripts. Transcript frequencies are shown on the left. (C) COL7A1 expression quantification by real-time qPCR using Taqman probes for two different COL7A1 regions (ex64, specific for all COL7A1 transcripts; and Ex80 probe, for exon 80-containing transcripts).

Article Snippet: Primary human RDEB and healthy donor keratinocytes were cultured as previously described., Human primary RDEB keratinocytes from patients P1 and P2 were plated onto lethally irradiated 3T3-J2 cells and cultured in keratinocyte growth complete FAD (cFAD) medium (KCa), a 3:1 mix of DMEM and Ham’s F12 medium (Gibco-BRL, Barcelona, Spain) containing fetal bovine calf serum (Hyclone, GE Healthcare, Logan, UT) (10%), penicillin-streptomycin (1%), glutamine (2%), insulin (5 μg/mL; Sigma-Aldrich), adenine (0.18 mmol/L; Sigma-Aldrich), hydrocortisone (0.4 μg/mL; Sigma-Aldrich), cholera toxin (0.1 nmol/L; Sigma-Aldrich), triiodothyronine (2 nmol/L; Sigma-Aldrich), epidermal growth factor (EGF) (10 ng/mL; Sigma-Aldrich), and Y-27632 Rho-associated protein kinase (ROCK) inhibitor (Sigma-Aldrich) at 10 μM.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Produced, Modification, Molecular Weight, Marker, Negative Control, Sequencing

Journal: Molecular Therapy

Article Title: Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing

doi: 10.1016/j.ymthe.2019.03.007

Figure Lengend Snippet: Collagen VII Expression in ΔE80 RDEB Polyclonal Keratinocytes Keratinocytes were treated with the RNP complex containing different CRISPR dual sgRNA combinations, and C7 expression was assessed by immunofluorescence (IF) and western blot. (A) C7 IF analysis. Left top: control, untreated RDEB (c.6527insC) keratinocytes are shown. Right top: RDEB keratinocytes were treated with the sg2 + sg4 pair. Left bottom: RDEB keratinocytes were treated with the sg1 + sg3 pair. Right bottom: RDEB keratinocytes were treated with the sg2 + sg3 pair. DAPI was used to stain nuclei. Scale bars, 50 μm. (B) Western blot analysis of C7 expression in unedited and edited RDEB keratinocytes, showing C7 bands intensities consistent with the IF data. (C) Western blot analysis of secreted C7 from culture supernatant of normal, untreated RDEB and the sg2 + sg3 RNP-treated RDEB keratinocytes. Protein loading was assessed by Ponceau red staining of the membrane (bottom). The ΔE80 C7 in edited RDEB cells is indistinguishable from that of normal human keratinocytes.

Article Snippet: Primary human RDEB and healthy donor keratinocytes were cultured as previously described., Human primary RDEB keratinocytes from patients P1 and P2 were plated onto lethally irradiated 3T3-J2 cells and cultured in keratinocyte growth complete FAD (cFAD) medium (KCa), a 3:1 mix of DMEM and Ham’s F12 medium (Gibco-BRL, Barcelona, Spain) containing fetal bovine calf serum (Hyclone, GE Healthcare, Logan, UT) (10%), penicillin-streptomycin (1%), glutamine (2%), insulin (5 μg/mL; Sigma-Aldrich), adenine (0.18 mmol/L; Sigma-Aldrich), hydrocortisone (0.4 μg/mL; Sigma-Aldrich), cholera toxin (0.1 nmol/L; Sigma-Aldrich), triiodothyronine (2 nmol/L; Sigma-Aldrich), epidermal growth factor (EGF) (10 ng/mL; Sigma-Aldrich), and Y-27632 Rho-associated protein kinase (ROCK) inhibitor (Sigma-Aldrich) at 10 μM.

Techniques: Expressing, CRISPR, Immunofluorescence, Western Blot, Staining

Journal: Molecular Therapy

Article Title: Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing

doi: 10.1016/j.ymthe.2019.03.007

Figure Lengend Snippet: Skin Regeneration, Collagen VII Expression, and Ultrastructural Analysis of Grafts from CRISPR/Cas9-Edited (ΔE80) Polyclonal Keratinocytes (A and B) Macroscopic view of engrafted areas 12 weeks after grafting of bioengineered skins containing gene-edited (ΔE80) (A) or unedited RDEB keratinocytes (B). (C and D) Histological analysis (H&E staining) of grafts from gene-edited (ΔE80) (C) or unedited RDEB keratinocytes (D). The dermal-epidermal separation in grafts from control, unedited cells is indicated by the red asterisk. (E and F) Human involucrin (h-inv) immunostaining (suprabasal expression) showing normal epidermal differentiation in grafts from ΔE80 keratinocytes (E) or unedited RDEB keratinocytes (F). (G and H) C7 immunoperoxidase expression analysis showing the continuous and correct deposition of C7 at the BMZ in ΔE80-edited grafts (G) and its complete absence in unedited RDEB keratinocyte grafts (H). Scale bars, 100 μm. (I and J) Electron microscopy analysis shows the presence of mature anchoring fibrils (arrows) at the dermal-epidermal junction of the gene-edited RDEB skin (I) and an empty electron lucent split area, corresponding to a blister (red asterisk) in the unedited graft (J). Scale bars, 200 nm (I) and 6 μm (J).

Article Snippet: Primary human RDEB and healthy donor keratinocytes were cultured as previously described., Human primary RDEB keratinocytes from patients P1 and P2 were plated onto lethally irradiated 3T3-J2 cells and cultured in keratinocyte growth complete FAD (cFAD) medium (KCa), a 3:1 mix of DMEM and Ham’s F12 medium (Gibco-BRL, Barcelona, Spain) containing fetal bovine calf serum (Hyclone, GE Healthcare, Logan, UT) (10%), penicillin-streptomycin (1%), glutamine (2%), insulin (5 μg/mL; Sigma-Aldrich), adenine (0.18 mmol/L; Sigma-Aldrich), hydrocortisone (0.4 μg/mL; Sigma-Aldrich), cholera toxin (0.1 nmol/L; Sigma-Aldrich), triiodothyronine (2 nmol/L; Sigma-Aldrich), epidermal growth factor (EGF) (10 ng/mL; Sigma-Aldrich), and Y-27632 Rho-associated protein kinase (ROCK) inhibitor (Sigma-Aldrich) at 10 μM.

Techniques: Expressing, CRISPR, Staining, Immunostaining, Electron Microscopy

Journal: Molecular Therapy

Article Title: Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing

doi: 10.1016/j.ymthe.2019.03.007

Figure Lengend Snippet: Skin Regeneration, C7 Expression, and Ultrastructural Analysis of Grafts from CRISPR/Cas9-Edited (ΔE80) Stem Cell Clones Keratinocyte clones, derived from the polyclonal population of cells treated with the sg2 + sg3 combination, with monoallelic (ΔE80/mut) and biallelic (ΔE80/ΔE80) E80 deletions being used to generate bioengineered skin equivalents and grafted onto immunodeficient mice. (A and B) Top: macroscopic view of an engrafted mouse 20 weeks after grafting of ΔE80/mut clone (A) and ΔE80/ΔE80 clone (B). Bottom: close-up view of the grafts under white and blue (GFP) light show the engrafted areas. (C and D) Histological analysis (H&E) of regenerated skin from ΔE80/mut clone (C) and ΔE80/ΔE80 clone (D). Note the clear dermal-epidermal adhesion in both types of grafts. (E and F) C7 immunoperoxidase expression analysis in ΔE80/mut clone (E) and ΔE80/ΔE80 clone (F) grafts. Both types of grafts display robust and continuous C7 expression. Insets show C7 expression at the human-mouse skin boundary. Note that mouse C7 is not recognized at the dilution of the antibody used. Scale bars, 100 μm. (G and H) Electron microscopy analysis shows the presence of well-developed anchoring fibrils (arrowheads) at the dermal-epidermal junction in grafts from both types of gene-edited clones. Scale bars, 500 nm.

Article Snippet: Primary human RDEB and healthy donor keratinocytes were cultured as previously described., Human primary RDEB keratinocytes from patients P1 and P2 were plated onto lethally irradiated 3T3-J2 cells and cultured in keratinocyte growth complete FAD (cFAD) medium (KCa), a 3:1 mix of DMEM and Ham’s F12 medium (Gibco-BRL, Barcelona, Spain) containing fetal bovine calf serum (Hyclone, GE Healthcare, Logan, UT) (10%), penicillin-streptomycin (1%), glutamine (2%), insulin (5 μg/mL; Sigma-Aldrich), adenine (0.18 mmol/L; Sigma-Aldrich), hydrocortisone (0.4 μg/mL; Sigma-Aldrich), cholera toxin (0.1 nmol/L; Sigma-Aldrich), triiodothyronine (2 nmol/L; Sigma-Aldrich), epidermal growth factor (EGF) (10 ng/mL; Sigma-Aldrich), and Y-27632 Rho-associated protein kinase (ROCK) inhibitor (Sigma-Aldrich) at 10 μM.

Techniques: Expressing, CRISPR, Clone Assay, Derivative Assay, Electron Microscopy