Transient puromycin selection increases reporter KI efficiency.

(A) Schematic of gene reporter design created by CRISPR editing. (B) Phase and fluorescence microscopy images of TBP-P2A-eGFP KI into H9 hESCs after puromycin selection. Scale bar = 100 μm. (C) Representative images of flow cytometry assessment of TBP-P2A-eGFP KI into H9 hESCs with and without transient puromycin selection. Untransfected cells were used to set the gates for reporter negative cells. Two peaks of eGFP+ cells can be observed in the puromycin treated group suggesting homozygous and heterozygous KI. (D) Flow cytometry analysis of TBP-P2A-eGFP, MYC-P2A-eGFP, and SOX2-P2A-eGFP KI into H9 hESCs with and without transient puromycin selection. For TBP n = 2 for both groups, for MYC n = 2 for replicates without puromycin and n = 5 for replicates with puromycin treatment, for SOX2 n = 3 for replicates without puromycin and n = 4 for replicates with puromycin treatment. n = biological replicates. p values: TBP = 0.0047, MYC = 0.0041, SOX2 = 0.0057. ** = p < .01. Unpaired two tailed t-test was used.</p

Dual KI of <i>TBP</i> and <i>MYC</i> or <i>TBP</i> and <i>SOX2</i> fluorescent reporters into H9 hESCs.

(A) Representative images of flow cytometry assessment of dual KI of TBP-P2A-eGFP and MYC-P2A-tdTomato or TBP-P2A-tdTomato and SOX2-P2A-eGFP into H9 hESCs with and without transient puromycin selection. (B) Flow cytometry analysis of the dual KI described in A. n = 2 for both dual KI combinations. n = biological replicates. (C) Fluorescence and brightfield microscopy of H9 hESCs positive for both reporter genes. Scale bar = 400 μm.</p

Demonstration of increased reporter KI efficiency in multiple human PSC lines after transient puromycin treatment.

(A) Representative images of flow cytometry assessment of SOX2-P2A-eGFP KI into EP1 hiPSCs with and without transient puromycin selection followed by flow cytometry analysis. Untransfected cells were used to set the gates for reporter negative cells. n = 2 for both groups. p value = 0.0039. (B) Flow cytometry analysis of TBP-P2A-eGFP KI into EP1 hiPSC, H7 hESC, and IMR90-4 hiPSC lines, respectively, with and without transient puromycin selection. For EP1 and H7, n = 2 for both groups, for IMR90-4 n = 3 for replicates without puromycin and n = 4 for replicates with puromycin treatment. n = biological replicates. p values: EP1 = 0.0246, H7 = 0.1532, IMR90-4 = <0.0001. * = p < .05, ** = p < .01, **** = p < .0001. ns = not significant. Unpaired two tailed t-test was used.</p

Zeocin replacement of puromycin results in similar KI efficiency of fluorescent reporter genes into human PSCs.

(A) Representative images of flow cytometry assessment of TBP-P2A-eGFP KI into IMR90-4 hiPSCs and H7 hESCs with and without transient zeocin selection. (B) Flow cytometry analysis of TBP-P2A-eGFP KI in part A. For IMR90-4, n = 3 for replicates without zeocin and n = 4 for replicates with zeocin treatment, for H7 n = 2 for replicates without zeocin and n = 5 for replicates with zeocin treatment. n = biological replicates. p values: IMR90-4 = 0.0172, H7 = 0.0036; * = p p < .01; Unpaired two tailed t-test was used.</p

Transient puromycin selection results in high KI efficiency of fluorescence reporter genes into mESCs.

(A, B, C) PCR tests for fluorescent reporter KI at the indicated loci in mESCs. For A and B, primers amplifying a region inside the KI gene and outside the donor plasmid template were used. For C, primers spanning the integration region were used to distinguish between homozygous and heterozygous clones. Expected amplicon sizes are shown. WT = wildtype. For KI assessment in B, lanes 9 and 17 were not counted as positive KI because the amplicon did not run at the predicted size. (D) Phase and fluorescence microscopy of a homozygous Six6-P2A-eGFP reporter KI line generated in C. mESCs were differentiated to optic vesicles for 8 days. Scale bar = 275 μm.</p

Transfection efficiency of H9 hESCs using lipofection.

(A) Phase/brightfield and fluorescence microscopy images of eGFP or Cas9-mKate2 plasmid transfected H9 hESCs are shown. Scale bar = 100 μm. (B) Flow cytometry assessment of H9 hESCs transfection. Untransfected cells were used to set the negative gates. BSC-A = back scatter area. Cells were analyzed at 40 hours post transfection in both A and B.</p

Generation of an RGC reporter line in EP1 hiPSC background using transient puromycin treatment.

(A) PCR zygosity test for KI at the targeted BRN3B locus. Primers spanning the integration region were used to amplify genomic DNA from randomly picked colonies derived from plating single cells. Homozygous insertion of the KI cassette is indicated by a single band at 3.3 kilobase pairs (kb). KI negative clones generate a band of 1.3 kb. Clones producing both bands were scored as heterozygous KI. WT = wildtype. For some of the clones (e.g. lane 6), the KI product is split into two parts due to an incorporation of only one monomer of the tdTomato sequence. (B) Fluorescence and phase microscopy of a differentiated EP1 hiPSC RGC reporter line generated using transient puromycin selection. Cells were imaged on day 29 of differentiation. Scale bar = 1000 μm.</p

Specification and differentiation of the ocular adnexa at E15.5.

<p>Cryosections were stained with various markers of keratinocyte differentiation. For orientation purposes c  =  cornea and r  =  retina in all images. Keratin 10 (K10) is expressed in papebral epidermis (ep) in wild type (A, +/+) and LIMK2-deficient (B, −/−) eyelids. The conjunctiva (conj.) is negative for K10 expression. An antibody recognizing phosphorylated c-Jun labels nuclei in cells (arrows) at the tip of the eyelid in both +/+ (C) and −/− (D) mice. Keratin 6 (K6) is expressed in epithelial cells at the tip of the eyelids in both +/+ (E) and −/− (F) mice, consistent with induction of a migratory phenotype. Scale bar  = 100 μm.</p

Phalloidin staining of filamentous actin in primary keratinocytes <i>in vitro</i> and developing eyelids <i>in vivo</i>.

<p>Primary keratinocytes were isolated from newborn pups and cultured in either low (60 μM; A, B) or high (1.2 mM; C, D) Ca²⁺ to induce differentiation. (A) Abundant stress fibers are apparent in wild type (+/+) keratinocytes. However, the intensity of phalloidin staining is decreased in LIMK2-deficient keratinocytes (B, −/−). The micrographs in A and B were taken at equal exposures. (C) After stimulation with Ca²⁺ for 24 hours, prominent cortical actin and stress fibers are present in +/+ keratinocytes. (D) Although the overall cell morphology in LIMK2-deficient keratinocytes is comparable to wild type cells, the intensity of cortical phalloidin stain is decreased. (E) Confocal microscopy of wholemount E15.5 ocular adnexa stained with phalloidin reveals intense stress fibers in the +/+ epithelium (ep) adjacent to the basal layer (bl). Cortical actin is also evident in the leading periderm cells (p) in the migrating tip. (F) Although cortical actin is obvious in the −/− periderm cells, stress fibers were not readily apparent in the adjacent epithelium. Scale bar  = 50 μm in A–D and 75 μm in E and F.</p

Ocular anatomy prior to eyelid fusion.

<p>H & E-stained paraffin sections of eyelids obtained from E14.5 (A and B) and E15.5 (C and D) embryos. No phenotypic differences are apparent in <i>Limk2</i> knockout (B) mice compared with control (A). The eyelid of both heterozygous (+/−) and homozygous (−/−) is largely comprised of mesenchyme (m) covered by epithelium (ep). The morphology of periderm (p) cells in the eyelid tip is round in contrast to the flat on the surface of the eyelid. Arrows in A and B point to specialized periderm cells that have changed from a squamous to a rounded morphology in preparation for epithelial sheet migration. A phenotype is clearly observed in E15.5 specimens. (C) The epithelium (ep) in heterozygous mice (+/−) has matured into a sheet of cells behind the rounded peridermal cells (p) that lead the migrating front across the cornea. The morphology of epithelial cells in the basal layer (bl) is clearly distinct from the adjacent mesenchymal cells (m) and the superficial epithelial cells. (D) Although rounded peridermal (p) cells are present in the <i>Limk2</i>-deficient eyelids, there is no evidence of epithelial sheet extension across the cornea. Scale bar  = 50 μm.</p