For CRISPR/Cas9-mediated cleavage of the first exon or 3’ end of porcine PTF1A, the following sgRNAs were selected (5’end targeting: 5’-gtcctcttactttgacgagg-3’; 3’end targeting: 5’-acataaccagatcctcagga-3’) and cloned into a pX462-Cas9^D10A nickase (Cas9n; D10A variant) (5’end) or pX330-Cas9 vector (3’end) carrying a puromycin resistance. For the generation of the targeting vectors, the homology arms were amplified by PCR from genomic DNA of German landrace pigs. The 5’ targeting vector/donor DNA consists of 1053 bp 5’ and 1246 bp 3’ homology arms (HA) flanking the iCre-polyA sequence. The 3’ donor DNA consists of 900 bp 5’ and 3’ HAs, a T2A self-cleaving peptide sequence, and the iCre-polyA sequence. To increase the efficiency of homology-directed repair (HDR) and the associated knock-in of iCre, the homology arms were flanked by the respective sgRNA target sequence in both targeting vectors, leading to the generation of double cut donors (11) ( Figure 1A ).

Porcine adipose-derived mesenchymal stem cells (ADMSCs) and porcine kidney fibroblast cells (KFCs) were isolated from a male German Landrace pig by the standard method (12). ADMSCs and KFCs were cultured with high-glucose DMEM supplemented with 10% FBS, 2 mM NEAA, 2 mM L-glutamine. Cells were passaged every 3-5 days and maintained at 50-90% confluency in an incubator at 37°C and 5% CO₂. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Passage 0 KFCs were co-transfected with pX462-Cas9^D10A and 5’ donor DNA vectors for targeting into exon 1 and passage 1-2 ADMSCs with pX330-Cas9 and 3’ donor DNA vectors for iCre placement into 3’ end of one PTF1A allele. Transfection was performed by electroporation of 1 x 10⁶ cells using 300V and 2 pulses (BTX ECM 830, Electro Square Porator). Following plasmid concentrations were used for transfection: 5’end targeting: 1.5 µg pX462-CRISPR/Cas9n and 4 µg 5’ double cut donor DNA; 3’end: 1 µg pX330-CRISPR/Cas9 and 4 µg 3’ double cut donor DNA. One day after transfection, the cells were selected for 48 h with 1.5 µg/ml (5’end targeting) and 3 µg/ml puromycin (3’ end targeting) and then expanded and analysed as single cell clones.

DNA isolated from individual stable transfected cell clones or founder animals was used for targeting PCR across the 5’ and 3’ junctions of the targeted PTF1A allele using the following primers for a) 5’end targeting: targ_up_F (5’-ctctctggagcctggctttta-3’) and iCre_R (5’-agagtcatccttggcaccat-3’) for 5’ junction PCR; iCre_F (5’-caagctggtggagagatgga-3’) and targ_Ex2_R (5’-ctggccagagttgttccaac-3’) for 3’ junction PCR ( Figure 1A ). The amplified PCR products were 2233 bp for 5’ and 2346 bp for 3’ junction respectively ( Figure 1B ). b) 3’end targeting: targ_Ex1_F (5’-ctgcacgagtactgctaccg-3’) and iCre_R for 5’ junction PCR and iCre_F and targ_down_R (5’-gctgaaagggatgagagggt-3’) for 3’ junction PCR ( Figure 1A ). The PCR products were 2127 bp for 5’ and 2300 bp for 3’ junction PCR respectively ( Figure 1B ). Correct insertion was confirmed by DNA sequencing of the PCR products.

Detection of a nontargeted PTF1A wild-type allele confirmed the monoallelic iCre insertion. For PCR amplification of the wild-type allele the following primers were used: targ_up_F and targ_Ex2_R for PTF1A^5’-iCre and targ_Ex1_F and targ_down_R for PTF1A^3’-iCre ( Figure 1A ), resulting in PCR products of 3020 bp and 2887 bp, respectively ( Figure 1B ). All PCRs were carried out with Phire Hot Start II DNA Polymerase (Thermo Fisher Scientific). Cycling thermal conditions were: 4 min, 98°C then 40 cycles of: 20 s, 98°C, 20 s, 65°C (for 5’ junction PCR), 63.6°C (3’ junction PCR), 64°C (for 5’ and 3’ endogenous PCR), 55 s, 72°C followed by 3 min 72°C.

Total RNA was isolated using InnuSPEED Tissue RNA Kit (Analytic Jena). After DNase digest (Turbo DNA-free™ Kit, Invitrogen), RNA was reverse transcribed using FastGene Scriptase II cDNA Kit (Nippon Genetics). RT-PCR was performed with Phire Hot Start II DNA Polymerase (Thermo Fisher Scientific) using forward primer hybridizing to exon 1 of PTF1A (PTF1A^5’-iCre : 5’-ctctcagcttcagcacatcg-3’, PTF1A^3’-iCre : 5’-gaaggtcatcatctgccacc-3’) and reverse primer to the iCre (5’-ggtcaaagtcagtgcgttca-3’), resulting in PCR products of 701 bp and 746 bp in length. Wildtype PTF1A mRNA expression was examined using primers hybridizing to exon 1 and 2 of PTF1A (5’-gaaggtcatcatctgccacc-3’ and 5’-ttgagtttcctggggtcctc-3’), resulting in a PCR product of 174 bp in length.

Off-targets sites for sgRNA for 5’ and 3’end targeting strategy were predicted with the use of CRISPOR (www.crispor.tefor.net) website tool. Five highest scoring potential off-target sites were analysed by PCR followed by sequncing.

Two selected cell clones for PTF1A^5’iCre targeting and three cell clones for PTF1A^3’iCre targeting were sent for SCNT. SCNT was performed as previously described (13).

Specimens were fixed in 4% formaldehyde solution for 48 h, embedded in paraffin, and sectioned (2 µm). After deparaffinization, antigen demasking (citrate buffer, pH=6) and inactivation of endogenous peroxidases (3% H₂O₂ in methanol), permeabilization was performed (1% goat serum, 0.05% Tween-20 and 0.4% Triton-X-100 in PBS) for 10 min. After blocking for 1 h (2% goat serum in PBS), sections were incubated overnight at 4°C with primary antibodies. Following antibodies were used: anti-Cre Recombinase in 1:100 dilution (D7L7L XP^® Rabbit mAb #15036, Cell Signaling). After 1 h incubation with Horseradish peroxidase (HRP)-conjugated secondary antibody (Goat Anti-Rabbit IgG-HRP #4030-05, SouthernBiotech) in 1:200 dilution, detection was performed using Vector DAB substrate (Vector Laboratories). Afterwards, the sections were counterstained with hematoxylin.

The pancreata were dissected, embedded in tissue freezing medium (Leica Biosystems), and sectioned at 12 μm. Tissue sections were fixed in pre-chilled 4% paraformaldehyde for 15 min at 4°C, then dehydrated in a progressive ethanol gradient at room temperature. Sections were permeabilized for 30 min at 40°C, blocked (10% FCS, 0.1% BSA, and 3% donkey serum in PBS with 0.1% Tween-20) for 1 h at room temperature (RT), and incubated with primary antibodies overnight at 4°C. Sections were washed in PBS, incubated with secondary antibodies (1:800 in blocking solution) for 3 h at 4°C, counterstained with DAPI for 20 min at RT, and mounted with the ProLong™ Diamond Antifade Mountant (ThermoFisher). Slides were dried overnight at RT and stored at 4°C until imaging by confocal microscope (ZEISS, LSM 880 with Airyscan). The following primary antibodies were used for immunofluorescent stainings: chicken anti-GFP (1:100, ThermoFisher); rat anti-RFP(1:500, Chromotek); rabbit anti-RFP (1:500, Rockland); rabbit anti-cytokeratin (1:500, Agilent); mouse anti-insulin (1:1000, Sigma); mouse anti-glucagon (1:500, Sigma); mouse anti-CPA1 (1:1000, LSBio). The following secondary antibodies were used: donkey anti-chicken Alexa Fluor 488 (Dianova); donkey anti-rat Cy3 (Dianova); donkey anti-rabbit Alexa Fluor 555 (ThermoFisher); donkey anti-mouse Cy5 (Dianova); donkey anti-rabbit AlexaFluor 649 (ThermoFisher).

To assess PTF1A-iCre expression in the newborn founder animals, tissue samples (pancreas, stomach, kidney, spleen, heart, muscle and lung) were collected for RT-PCR and immunohistochemical analyses. At this developmental stage iCre expression was restricted to pancreatic acinar and single gastric cells in both PTF1A^5’iCre and PTF1A^3’iCre targeted pigs ( Figures 1D , 2A, B ). The other organs tested were negative. These results are in accordance with PTF1A/p48 expression in humans (https://www.proteinatlas.org/ENSG00000168267-PTF1A/tissue).

To assess the location, pattern, and extent of iCre expression during fetal development and to prove iCre function, founder boars were crossed with a dual-fluorescence reporter pig line R26^mT/mG (19). This line carries a membrane-targeted tandem dimer GFP (mGFP) gene downstream of a floxed mTomato transgene at the porcine ROSA26 locus ( Figure 3A ). Cre mediated excision of mTomato results in a switch from red to green fluorescence (20), marking all cells during development in which Cre recombinase was active.

F1 animals showed near Mendelian inheritance: 3/8 (37.5%) for PTF1A^5’iCre and 4/11 (36.7%) for PTF1A^3’iCre . On postnatal day 1, one R26^mT/mGPTF1A^5’iCre and one R26^mT/mGPTF1A^3’iCre piglet were sacrificed, organs isolated, and fluorescent imaging carried out. Figure 3B shows extensive green fluorescence in the pancreas and cerebellum and single positive cells in the stomach. Other tissues such as the kidney were negative.

Focusing on the pancreas multi-color immunofluorescence staining was performed for cell type identification. As shown in Figure 4A mGFP is expressed in glucagon-producing α-cells, ductal cells, and acinar cells. Interestingly, for β-cells co-staining of insulin with both RFP and GFP was observed ( Figure 4B ). Colocalization of these pancreatic cell types with GFP confirmed the participation of PTF1A to all pancreatic lineages during development.