|Ahead of print publication
Spatiotemporal gene targeting in the mouse corneal endothelium
JeongGoo Lee, Martin Heur
USC Roski Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA
|Date of Submission||13-Sep-2022|
|Date of Acceptance||27-Oct-2022|
|Date of Web Publication||11-Jan-2023|
USC Roski Eye Institute, 1450 San Pablo, Ste 4900, Los Angeles 90033, California
Source of Support: None, Conflict of Interest: None
PURPOSE: The inducible Cre-ERT2 recombinase system allows for temporal control of gene targeting, and it is useful to studying adult function of genes that have critical developmental roles. The Zeb1flox/flox: UBC-CreERT2 mouse was generated to conditionally target Zeb1 to investigate its role in mesenchymal transition in the mouse corneal endothelium in vivo.
MATERIALS AND METHODS: Hemizygous UBC-CreERT2 mice were crossed with homozygous mice harboring loxP-flanked Zeb1 alleles (Zeb1flox/flox) to generate the Zeb1flox/flox: UBC-CreERT2 mouse. 4-hydroxytamoxifen (4-OHT) exposure leads to excision of exon 6 of Zeb1, resulting in a loss function allele in the Zeb1flox/flox: UBC-CreERT2 mouse. Intracameral 4-OHT injection further isolates Zeb1 targeting to the anterior chamber. Mesenchymal transition and induction of Zeb1 expression in the corneal endothelium was achieved using FGF2 in ex vivo organ culture. Gene expression was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and by immunoblotting in the mouse corneal endothelium in vivo.
RESULTS: Following Cre-mediated targeting of Zeb1 by intracameral 4-OHT injection in Zeb1flox/flox: UBC-CreERT2 mice, FGF2 treatment in ex vivo organ culture resulted in abrogation of Zeb1 mRNA and protein expression in the corneal endothelium.
CONCLUSION: The data show Zeb1, a critical mediator of fibrosis in corneal endothelial mesenchymal transition, can be targeted by intracameral injection of 4-OHT in the mouse corneal endothelium in vivo. These results suggest that genes with critical developmental roles can be targeted at a specific time in the corneal endothelium to study its role in adult disease using an inducible Cre-Lox strategy.
Keywords: Conditional targeting, corneal endothelium, mouse
| Introduction|| |
Conditional gene targeting is a powerful technique that allows for studying gene function in one tissue without affecting expression in other tissues. This is particularly useful for studying adult function of a gene if knocking it out of the whole organism leads to an embryonic or perinatal lethal phenotype. Tissue-specific activation of Cre recombinase (Cre) paired with flanking a gene of interest with loxP sequences can be used to avoid the above-mentioned limitations observed in some constitutive knockout models. Furthermore, temporal control of Cre activation can prevent potential developmental impact of constitutively knocking out a gene.,, The Cre recognizes two loxP sequences and mediates excision of the intervening DNA sequence and allows for gene targeting in specific tissue and specific time using an inducer such as 4-hydroxytamoxifen (4-OHT).,, The second generation of tamoxifen inducible- Cre is composed of Cre fused with a mutant human estrogen receptor harboring a G400V/M543A/L544A triple mutation in the ligand binding domain (ERT2) which is more sensitive and specific for 4-OHT than the wildtype estrogen receptor.,, The fusion protein is normally located in the cytoplasm bound to heat shock protein 90 (HSP90). Binding of 4-OHT to CreERT2 leads to its release from HSP90, translocation into the nucleus, and excision of the target sequence between the loxP sites.,,
Cell proliferation, migration, and fibrosis, critical for restoring tissue integrity in wound healing, are features observed in mesenchymal transition. Mesenchymal transition is a process where cells lose their polarity, assume a fibroblastic phenotype, and exhibit enhanced cell proliferation, migration, and type I collagen secretion, and corneal endothelial cells (CECs) can undergo mesenchymal transition (EnMT) in response to severe injury or inflammation.,,, Unlike other tissues where restoration of tissue integrity is beneficial, EnMT-induced fibrosis leads to retrocorneal membrane (RCM) formation with subsequent vision loss in the cornea and is undesirable. Zinc finger E-box binding homeobox 1 (Zeb1) plays a critical role in induction of fibrosis in EnMT in human and mouse corneal endothelium ex vivo. Surgical injury can induce FGF2 expression and EnMT in the mouse corneal endothelium in vivo, and siRNA-mediated knockdown of Zeb1 can inhibit injury-dependent RCM formation in the mouse corneal endothelium in vivo.,
Although targeting Zeb1 by siRNA or blocking its interaction YAP1 with verteporfin or CtBP by NSC95397 and MTOB is possible, these approaches are fraught by non-specific or off target effects., A genetic approach to targeting Zeb1 is more desirable, but the Zeb1 null mouse cannot be used to study Zeb1 function in EnMT in the adult corneal endothelium because knocking out Zeb1 leads to a perinatal lethal phenotype due to numerous developmental abnormalities. These limitations led us to generate the Zeb1flox/flox: UBC-CreERT2 mouse to allow for temporal and spatial control of Zeb1 targeting. In the Zeb1flox/flox: UBC-CreERT2 mouse, CreERT2 is expressed under the control of Ubiquitin C promoter, and intracameral injection of 4-OHT allows for temporal targeting of Zeb1 in the mouse corneal endothelium in vivo. Here, we report that the Cre-loxP approach can be used to achieve conditional gene targeting in the mouse corneal endothelium in vivo.
| Materials and Methods|| |
All mouse experiments were performed in accordance with the protocol approved by University of Southern California Institutional Animal Care and Use Committee (IACUC protocol number 21213). The mice were housed in clear, air-filtered cages with 12-h light/dark cycle and ad lib feeding. C57BL/6 and C57BL/6 UBC-CreERT2 mouse breeding pairs were purchased from Jackson Laboratories (Sacramento, CA, USA) and C57BL/6 Zeb1flox/flox was acquired from Prof. Marc Stemmler. Zeb1flox/flox: UBC-CreERT2 mice were generated by crossing Zeb1flox/flox mice with UBC-CreERT2 mice. Colonies used in this study were bred in-house. Mice between ages of 12 and 14 weeks were used for all experiments. Mice were anesthetized by intraperitoneal injection of ketamine (60–70 mg/kg) and xylazine (5–10 mg/kg), and they were euthanized by cervical dislocation.
FGF2 was purchased from Cell Signaling Technology (Danvers, MA, USA). 4-hydroxytamoxifen was obtained from Tocris Bioscience (Minneapolis, MN, USA). Anti-β-actin (42 kDa, A5316) and peroxidase-conjugated secondary antibodies were obtained from MilliporeSigma (Burlington, MA, USA). Anti-ZEB1 (124 kDa, PA5–40350) antibody was purchased from Thermo Fisher (Waltham, MA, USA).
Genomic DNA extraction and amplification with gene-specific primers were performed using Phire Tissue Direct polymerase chain reaction (PCR) master mix (ThermoFisher Scientific). Primers for the detection of Cre-ERT2 (475bp): 5'-GACCTGACCCGTTCTGTTG-3' (forward) and 5'-AGGCAAATTTTGGTGTACGG-3' (reverse). Primers for the detection of Zeb1+/+, Zeb1flox/flox and Zeb1del/del alleles: Zeb1 F1, 5'-CGTGATGGAGCCAGAATCTGACCCC-3'; Zeb1 R1, 5'-GCCCTGTCTTTCTCAGCAGTGTGG-3'; Zeb1 F2, 5'-GTCACTTCTACACTGGCAGCTA-3'; Zeb1 R2, 5'-GCCATCTCACCAGCCCTTACTGTGC-3'. PCR conditions for genotyping were as follows: 2 min at 94°C, followed first by 20 s at 94°C, 15 s at 65°C, 10 s at 68°C for 10 cycles, then by 15 s at 94°C, 60 s at 15°C, 10 s at 72°C for 28 cycles, and with a final extension for 4 min at 72°C. DNA fragments were separated by electrophoresis on a 1.5% agarose gel to access whether the deletion of Zeb1 exon 6 and the presence of CreERT2 and Zeb1flox/flox. The animals used in this study were UBC-CreERT2 hemizygotes and Zeb1flox/flox homozygotes.
4-hydroxytamoxifen preparation and treatment
4-OHT was dissolved in 100% ethanol (molecular biology grade) at 20 mg/ml and diluted with corn oil (MilliporeSigma) for final concentration 10 uM. For intracameral injections, 3 μl of aqueous humor was aspirated and 3 μl of 10 μM 4-HOT was injected into the anterior chamber at the limbus using a 30-gauge needle daily for three consecutive days. The vehicle-injected contralateral eye was used as control. Mice were euthanized 7 days' post first intracameral injection of 4-OHT, and their eyes were enucleated. All procedures were performed under direct visualization using an operating microscope, and care was taken not to injure the lens. Corneas were excised from the enucleated eyes, the endothelium was stripped from the excised corneas, and the stripped endothelium was then processed for reverse transcription (RT)-PCR and immunoblotting.
Semi-quantitative reverse transcription-polymerase chain reaction analysis
The corneas were isolated from eyes of 6 mice per experimental group and the corneal endothelium-Descemet membrane complex was stripped using jeweler's forceps under a dissecting microscope. Total RNA was extracted from mouse corneal endothelium and RT-PCR was performed as previously described. Briefly, cDNA was synthesized with 0.5 μg of RNA by utilizing iScript reverse transcriptase (Bio-Rad, Hercules, CA, USA) and oligo (dT) primer. Reverse transcription was performed at 42°C for 90 min. Then, the first strand cDNA equivalent to 0.05 μg of starting RNA from each sample was amplified using the specific primer pairs. The specific primers and PCR conditions used are shown in [Table 1]. Standard PCR conditions were as follows: 5 min at 94°C, followed by 30 s at 94°C, 30 s at 53°C, 30 s at 72°C, and a final extension for 4 min at 72°C. PCR cycles were optimized to ensure that the product amplification fell within the linear phase of amplification and annealing temperature were adjusted depending on the PCR primers [Table 1]. RT-PCR amplification of β-actin transcript was used as loading control. The amplified products were separated on a 1.5% agarose gel electrophoresis, visualized by Gel-Red staining, and the band intensity was analyzed using Image Lab program from Bio-Rad. All target PCR products were verified by DNA sequencing.
|Table 1: Forward and reverse primer sequences, annealing temperature, and number of cycles performed for reverse transcription-polymerase chain reaction|
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Protein preparation, protein assay, SDS PAGE, and immunoblotting analysis
All assays were performed following previously reported protocols., The following gel concentrations were used to separate proteins: 10% polyacrylamide gel for β-actin and 8% polyacrylamide gel for ZEB1. The corneas were isolated from eyes of 18 mice per experimental group and the corneal endothelium-Descemet membrane complex was stripped using jeweler's forceps under a dissecting microscope. For protein purification from ex vivo corneal endothelium, cells in the tissue were lysed with RIPA lysis buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) in situ. Total protein was purified and concentrated with Amicon ultra centrifugal filter devices (MilliporeSigma), according to the manufacturer's instructions. Briefly, cell lysates were applied to the Amicon ultra 10 K centrifugal device (molecular weight cutoff 10 K), and then spin down at 14,000 × g for 30 min. To recover the concentrated protein, the Amicon ultra filter device placed upside down in a clean tube which was then centrifuged again for 2 min at 1000 × g to transfer the concentrated protein from the device to the clean tube. Purified total proteins were used to for analysis of immunoblotting.
| Results|| |
Generation of Zeb1flox/flox: UBC-CreERT2 mice
Mice harboring 2 alleles the Zeb1 gene with exon 6 flanked by loxP sequences (Zeb1flox/flox) were crossed to hemizygous UBC-CreERT2 transgenic mice to generate Zeb1flox/flox: UBC-CreERT2 mice [Figure 1]a. To induce nuclear translocation of the CreERT2 fusion protein and excision of exon 6,,, 4-OHT was injected in the anterior chamber of Zeb1flox/flox: UBC-CreERT2 and Zeb1flox/flox mice. Seven days after intracameral injections of 4-OHT or vehicle, genotyping was performed on purified genomic DNA isolated from corneal endothelium of Zeb1flox/flox: UBC-CreERT2 and Zeb1flox/flox mice. In the corneal endothelium of mice that received vehicle injections, PCR products of 295 bp with the F1-R1 primer pair and 512 bp with the F2-R2 primer pair were detected, while the 367 bp product with the F1-R2 primer pair was not detected [Figure 1]b. The corneal endothelium from Zeb1flox/flox: UBC-CreERT2 mice injected with 4-OHT showed the 367 bp product with F1-R2 primer pair, while no PCR products using F1-R1 and F2-R2 primer pairs were detected [Figure 1]b. Zeb1flox/flox mice injected with 4-OHT showed the same PCR product profile as the vehicle-injected mice.
|Figure 1: Schematic illustration of the Zeb1 flox allele and deletion of exon 6. (a) Gray boxes indicate exons, and triangles indicate the location of the loxP sites flanking exon 6. The location of F1, F2, R1, and R2 genotyping primers are shown by arrows. Cre recombinase activity leads to excision of exon 6 along with R1 and F2 primer sites. (b) The loxP sites were confirmed via genotyping with pair of primers; F1-R1 for proximal site and F2-R2 for distal site. Seven days after intracameral 4-OHT (+) or vehicle (−) injection, the endothelium was isolated and genomic DNA was purified for PCR genotyping. Exon 6 of Zeb1 was deleted in the corneal endothelium following intracameral 4-OHT injection in Zeb1flox/flox: UBC-CreERT2 mice but not in the other mice. In Zeb1flox/flox: UBC-CreERT2 mice that received intracameral 4-OHT injection, PCR genotyping with F1-R2 primer pair generated the expected 367 bp product while F1-R1 and F2-R2 reactions did not generate any products due to the loss of R2 and F1 sites following Cre-mediated recombination. In all other groups, F1-R1 and F2-R2 reactions generated the expected 295 bp and 512 bp products respectively while F1-R2 reaction did not generate the expected 367 bp product, indicating Zeb1 was not altered|
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Inhibition of FGF2-dependnet Zeb1 expression by intracameral injection of 4-OHT in the Zeb1flox/flox: UBC-CreERT2 mouse corneal endothelium
RT-PCR and immunoblotting was performed to determine the effect of exon 6 deletion on Zeb1 expression. Seven days after intracameral 4-OHT injections, corneas from wildtype, CreERT2 transgenic, Zeb1flox/flox, and Zeb1flox/flox: UBC-CreERT2 mice were excised and incubated with FGF2 for 7 days in organ culture to induce Zeb1 expression in the endothelium ex vivo. Zeb1 RT-PCR using a primer pair, in which the forward primer is located in exon 6 and the reverse primer is located in exon 7, shows that intracameral 4-OHT injection followed by FGF2 treatment in Zeb1flox/flox: UBC-CreERT2 mice led to the excision of exon 6 in the Zeb1 mRNA purified from the corneal endothelium [Figure 2]a. Intracameral 4-OHT injection followed by FGF2 treatment in wildtype, CreERT2 transgenic, and Zeb1flox/flox mice did not lead to excision of exon 6 in the Zeb1 mRNA purified from the corneal endothelium [Figure 2]a. FGF2 treatment led to an induction of Zeb1 mRNA expression in all mice, while treatment with vehicle did not induce Zeb1 mRNA expression. Col8A2 was used as a marker for CECs, beta-actin was used as a loading control, and Keratocan was used as a marker for stromal keratocytes [Figure 2]a. Excision of exon 6 leads to a truncated Zeb1 mRNA with a premature stop codon, and this led to an absence of Zeb1 protein in the corneal endothelium of Zeb1flox/flox: UBC-CreERT2 mice that received intracameral 4-OHT injection followed by FGF2 treatment [Figure 2]b. Zeb1 protein could be detected in the corneal endothelium of wildtype, CreERT2 transgenic, and Zeb1flox/flox mice that received 4-OHT injection and FGF2 treatment [Figure 2]b. β-actin was used as loading control.
|Figure 2: Inhibition of Zeb1 expression by intracameral 4-OHT injection in the corneal endothelium of Zeb1flox/flox: UBC-CreERT2 mice. (a) Seven days after intracameral 4-OHT (+) or vehicle (−) injections, the corneal endothelium from wildtype (WT), UBC-CreERT2, Zeb1flox/flox, and Zeb1flox/flox: UBC-CreERT2 mice, 6 mice per group, were isolated and maintained in organ culture with FGF2 or vehicle (Con) for 7 days ex vivo. Total RNA was isolated from the corneal endothelium and RT-PCR was performed for Zeb1, Col8a2, ActB, and Ktcn. FGF2 but not vehicle treatment induced Zeb1 expression in all groups as probed by using the forward primer located in exon 6 and the reverse primer located in exon 7. Intracameral injection of 4-OHT in Zeb1flox/flox: UBC-CreERT2 led to loss of exon 6 in the Zeb1 mRNA in the corneal endothelium. Col8a2 and Actb were used as CEC marker and loading control, respectively. Keratocan (Ktcn) was used to control for possible stromal keratocyte and epithelial cell contamination. (b) Seven days after intracameral 4-OHT (+) or vehicle (−) injections, the corneal endothelium from wildtype (WT), UBC-CreERT2, Zeb1flox/flox, and Zeb1flox/flox: UBC-CreERT2 mice, 18 mice per group, were isolated and maintained in organ culture with FGF2 or vehicle (Con) for 7 days ex vivo. Total protein was purified from the corneal endothelium and immunoblotting for Zeb1 and β-actin was performed. FGF2 but not vehicle treatment induced Zeb1 expression in the corneal endothelium of all groups except Zeb1flox/flox: UBC-CreERT2 mice that received intracameral 4-OHT injection. Zeb1 protein was not detectable in the corneal endothelium of Zeb1flox/flox: UBC-CreERT2 mice that received intracameral 4-OHT injection indicating targeting of Zeb1 in the corneal endothelium following 4-OHT injection|
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| Discussion|| |
The cornea is the anterior transparent tissue of the eye that serves as its main refractive element. Maintenance of transparency is critical for the refractive function, and it is dependent on the coordinated function of its layers, the epithelium, stroma, and endothelium. The corneal endothelium functions as a pump function and regulates corneal hydration, which is critical for maintenance of corneal transparency. Adult human CECs are G1 arrested but can undergo mesenchymal transition (EnMT) in response to severe injury., Endothelial cells that undergo EnMT show increased cell migration, proliferation and secrete collagen type I.,, The latter can lead to RCM formation, an opaque, fibrous membrane that can lead to irreversible blindness. Zeb1, a critical mediator of mesenchymal transition in many biological processes, was also identified to be a critical regulator of fibrosis in EnMT. Although siRNA knockdown of Zeb1 led to decreased RCM formation in the mouse corneal endothelium in vivo, a genetic approach to targeting Zeb1 is needed to validate the siRNA knockdown results due to potential siRNA off-target effects. The Zeb1 null mouse develops to term but shows severe defects in neural crest-derived skeletal elements including numerous craniofacial abnormalities, fused ribs, and defects of the sternum, and the embryo does not survive past the postnatal period. The role of Zeb1 in EnMT in the adult mouse cannot be studied using the Zeb1 null mouse because of its perinatal lethal phenotype. This led us to explore using conditional targeting as a genetic approach to studying the role of Zeb1 in EnMT in the adult mouse.
Zeb1flox/flox: UBC-CreERT2 mice were generated by crossing Zeb1flox/flox mice to UBC-CreERT2 transgenic mice to take advantage of the Cre-lox system for conditional targeting of Zeb1 in the corneal endothelium. Intracameral injection of 4-OHT in the Zeb1flox/flox: UBC-CreERT2 mouse led to excision of exon 6 in the genomic DNA of CECs in the adult mouse [Figure 1]b. Moreover, we did not observe spontaneous Cre activity in the corneal endothelium as evidenced by lack of the 367 bp F1-R2 PCR product in the Zeb1flox/flox: UBC-CreERT2 mice that received intracameral injection of vehicle only [Figure 1]b. Excision of exon 6 was not complete since a faint amount of the 512 bp product of F2-R2 reaction can be seen in the corneal endothelium of 4-OHT-injected Zeb1flox/flox: UBC-CreERT2 mice [Figure 1]b. We did not observe excision of exon 6 in the Zeb1flox/flox mice. The genomic modification in the corneal endothelium is reflected at the transcriptional level in the 4-OHT-injected Zeb1flox/flox: UBC-CreERT2 mice. FGF2 was used in organ culture to stimulate Zeb1 expression. Zeb1 RT-PCR using mRNA isolated from the corneal endothelium from 4-OHT-injected Zeb1flox/flox: UBC-CreERT2 mice showed a severe decrease in the amount intact Zeb1 mRNA [Figure 2]a. This likely reflects the small amount of intact Zeb1 gene in the corneal endothelium, and this could be due to an insufficient dosage of 4-OHT in the intracameral injection. Intracameral 4-OHT injection and FGF2 treatment induced Zeb1 mRNA expression in wildtype, UBC-CreERT2, and Zeb1flox/flox mice [Figure 2]a. A concern that arises from the RT-PCR results is whether the small amount of intact Zeb1 mRNA would lead to the presence of Zeb1 protein in the corneal endothelium of 4-OHT-injected Zeb1flox/flox: UBC-CreERT2 mice. Immunoblotting using proteins isolated from the corneal endothelium shows that Zeb1 could not be detected in of 4-OHT-injected Zeb1flox/flox: UBC-CreERT2 mice whose corneas were treated with FGF2 [Figure 2]b. Intracameral 4-OHT injection and FGF2 treatment induced Zeb1 protein expression in wildtype, UBC-CreERT2, and Zeb1flox/flox mice [Figure 2]b.
Our results show that genes in the corneal endothelium can be targeted in a conditional manner. This opens the door for studying adult function of genes in the corneal endothelium that have critical development roles, where knocking out the genes leads to an embryonic or perinatal lethal phenotype. In this report, we demonstrate proof of principle for spatiotemporal gene targeting in the mouse corneal endothelium using an inducible Cre-Lox strategy.
This work was supported NIH/NEI P30EY029220 and an Unrestricted Grant from Research to Prevent Blindness, New York, NY. Martin Heur was a previous recipient of a Career Development Award from Research to Prevent Blindness. The Zeb1 was a generous gift from Marc Stemmler, Simone Brabletz, and Thomas Brabletz from the Institute of Experimental Medicine I, Nikolaus-Fiebiger Center for Molecular Medicine in Erlangen, Germany.
Financial support and sponsorship
NIH/NEI P30EY029220 and Research to Prevent Blindness, New York, NY.
Conflicts of interest
There are no conflicts of interest.
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