In vivo enrichment of busulfan-resistant germ cells for efficient production of transgenic avian models

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Experimental animals and animal care

The care and experimental use of chickens was approved by the Institute of Laboratory Animal Resources, Seoul National University. Chickens were maintained according to a standard management program at the University Animal Farm, Seoul National University, Korea. All procedures, including chicken maintenance, reproduction, and sample collection, were governed by standard operating protocols according to a standard management program at the University Animal Farm, Seoul National University and the Animal Genetic Engineering Laboratory at Seoul National University.

Construction of human MGSTII expression vector

Codons of the human microsomal glutathione-S-transferase II (MGSTII) gene were optimised for expression in the hen using the Gallus gallus codon database (https://www.kazusa.or.jp/codon). A codon-optimized human MGSTII gene were integrated into piggyBac TK NeoR CMV GFP FRT backbone vector from our previous study19. Briefly, codon-optimized MGSTII CDS inserted into backbone vector by using HindIII, NotI restriction enzymes. After that, CMV MGSTII cassette was cloned by PCR including XhoI restriction site at the 5′ end and 3′ end. This cloned cassette was integrated into piggyBac TK NeoR CMV GFP FRT backbone by using XhoI restriction enzyme and produce final vector piggyBac TK NeoR CMV GFP CMV MGSTII.

Transfection and G418-selection of PGCs

The methods of cultivation, transfection and G418-selection of WL male PGC cells were followed by our previous report17. Briefly, male PGCs were cultured on mitotically inactivated mouse fibroblast cells (MEFs) in knockout Dulbecco’s Modified Eagle’s Medium (KO-DMEM) (Invitrogen, Life Technologies, Carlsbad, CA, USA) supplemented with 20% (v/v) fetal bovine serum (Invitrogen, Life Technologies), 2% (v/v) chicken serum (Sigma-Aldrich, St. Louis, MO, USA), 1 × nucleoside mix (EMD Millipore, Temecula, CA, USA), 2 mM L-glutamine, 1 × nonessential amino acid mix, β-mercaptoethanol, 10 mM sodium pyruvate, 1 × antibiotic antimycotic mix (Invitrogen, Life Technologies), and human basic fibroblast growth factor (10 ng/mL; Koma Biotech, Seoul, Korea). PGCs were cultured at 37℃ in an atmosphere of 5% (v/v) CO2 and 60–70% relative humidity. The piggyBac TK NeoR CMV GFP CMV MGSTII expression vector and transposase (CAGG-PBase, pCyL43) were cotransfected into the PGC line using Lipofectamine 2000 (Invitrogen, Life Technologies). One day after transfection, G418 (300 mg/mL) was added to culture medium to enable selection of transfected PGCs for 1 month.

In vitro assay to assess the effect of busulfan resistance on MGSTII-expressing PGCs

WT and MGSTII-tg PGCs were incubated at 50,000 cells per well in 96-well tissue culture plates with varying concentrations of busulfan (1, 2, 4, 6, and 8 μM, Sigma-Aldrich) dissolved in dimethyl sulfoxide (DMSO). To assess the busulfan resistance of MGSTII-tg PGCs, cells were incubated in the specified concentrations of busulfan for 48 h. After incubation, cell viability was assessed using the WST-1 method (Roche Diagnostic, Basel, Switzerland). Briefly, WST-1 was added (15 μL/well) and cells were incubated for 4 h. The absorbance (A450-A650) of formazan dye produced by metabolically active cells was detected using an Epoch microplate reader (BioTek, Winooski, VT, USA).

Transplantation of MGSTII-tg PGCs into recipient embryos

To produce germline chimeric chickens through injection of MGSTII-tg PGCs into recipient embryos, we made a small hole at the pointed end of each recipient KO egg at Hamburger and Hamilton (HH) stages HH13–16, and microinjected a 2 μL aliquot containing at least 3,000 MGSTII-tg PGCs into the dorsal aortas of recipient embryos. Egg holes were sealed with paraffin film, and eggs were incubated with the pointed end down prior to further screening and eventual hatching. To assess the effect of busulfan on MGSTII-tg PGCs, approximately 1,000 GFP-tg PGCs, as generated in our previous report17, or MGSTII-tg PGCs were injected with 1 μM busulfan solution at HH13–16. Three days after transplantation, the gonads from a sample of the recipient embryos were collected at HH 28, and the presence of GFP-expressing PGCs in the gonads were imaged by fluorescence microscopy (Nikon) and manually counted.

Test-cross analysis and validation of transgenic chicken production efficiency

After sexual maturation, putative germline chimeras were testcrossed by mating with WL female chickens to generate transgenic chickens derived from transplanted donor PGCs. Transgenic (TG) chickens were identified using a fluorescent excitation lamp with detection filters (BLS Ltd., Budapest, Hungary), and were confirmed by genomic DNA PCR. To evaluate the donor-derived production efficiency and transgenic chick production efficiency, adult germline chimeras were injected with 40 mg/kg busulfan. Briefly, 40 mg busulfan dissolved in 1 mL N,N-dimethyl formamide (Merck, Darmstadt, Germany) was injected intraperitoneally into germline chimeras. Two weeks after busulfan injection, busulfan-treated germline chimeras were testcrossed by mating with wildtype WL female chickens. At least 20 progeny animals from both groups of germline chimeras (busulfan-treated or non-treated) were analyzed for fertilization rate and transgenic chick production efficiency by measuring hatching rates, fluorescence expression, and genotypes for 8 weeks.

PCR analysis and identification of the transgene integration site

To detect the presence of the integrated transgene and distinguish between the wild-type (WT) and MGSTII-tg loci, transgene specific primer sets (MGST F: 5′-CCA CCA TGG CAG GCA ACA GC-3′ and MGST R: 5′-CCG CTC AGA ATT GCC GCC TC-3′) were used. Genomic DNA PCR from WT and MGSTII-tg was also performed using GAPDH primers (F: 5′-GGT GGT GCT AAG CGT GTT AT-3′; R: 5′-ACC TCT GCC ATC TCT CCA CA-3′) as a control. Each PCR was performed in a total volume of 20 μL containing 100 ng genomic DNA from WT and MGSTII-tg, 10 × PCR buffer (BioFACT, Daejeon, Korea), 10 mM dNTPs, 5 pmol of each primer, and 0.5 U Taq polymerase (BioFACT) in the following thermocycling conditions: 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 60 °C, 30 s at 72 °C, and, finally, 10 min at 72 °C.

The transgene (piggyBac CMV GFP CMV MGSTII TK NeoR) insertion site was identified using the Genome Walker Kit (Takara, Japan) according to the manufacturer’s protocol and our previous report24. Gene-specific primers were designed from the known DNA sequence of the piggyBac transgene to move upstream of the gene in genomic DNA. Genomic DNA was digested with four different restriction enzymes (EcoRV, DraI, PvuII, and SspI) ligated with adaptors, and PCR was performed using the polymerase mix. PCR products were excised from agarose gel, purified using a Power Gel Extraction Kit (Promega, Madison, WI, USA), and subsequently cloned into the pGEM-T Easy Vector (Promega). Cloned PCR products were sequenced using an ABI Prism 3730 XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences of the 5′- flanking regions were analyzed using the Basic Local Alignment Search Tool (BLAST) Assembled Genome database (http://blast.ncbi.nlm.nih.gov/BLAST.cgi) and the UCSC Genome Bioinformatics browser (http://www.genome.ucsc.edu) to identify transgene integration sites in the transgenic chickens’ genomes.

Immunohistochemistry

The procedures of testis section and immunostaining were followed by our previous report47. Adult testes of WT chickens and busulfan-treated or untreated germline chimeric chickens were paraffin-embedded and sectioned (thickness, 10 μm). After deparaffinization, sections were washed three times with 1 × phosphate-buffered saline (PBS) and blocked with a blocking buffer (5% goat serum and 1% bovine serum albumin in PBS) for 1 h at room temperature. Sections were then incubated at 4 °C overnight with a rabbit anti-GFP primary antibody (Invitrogen, Carlsbad, CA, USA) (1:200 dilutions in blocking buffer). After washing three times with PBS, sections were incubated with fluorescence-conjugated secondary antibodies (Alexa Fluor 594 or 488, Invitrogen) for 1 h at room temperature. After washing three times with PBS, sections were mounted with Prolong Gold antifade reagent with DAPI and imaged using a confocal fluorescence microscope (Carl Zeiss Inc, Oberkocken, Germany).

Statistical analyses

To analyze dose-dependent effects of busulfan on MGSTII PGCs and WT PGCs, a two-way ANOVA was used to determine statistical significance. Significant differences between busulfan-treated and busulfan non-treated groups were examined using a one-way ANOVA. P < 0.05 was considered indicative of statistical significance. ***p < 0.001, **p < 0.01, *p < 0.05.

Ethics statement

All experimental procedures and care of chickens was approved by the Institute of Laboratory Animal Resources, Seoul National University, and all methods were carried out in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and approved by the Institutional Animal Care and Use Committee (IACUC, SNU‐190,401‐1–1) of Seoul National University, Korea.

Approval for animal experiments

All experimental procedures and care of chickens was approved by the Institute of Laboratory Animal Resources, Seoul National University (SNU‐190,401‐1–1), and all methods were carried out in accordance with guidelines and regulations of the Institutional Animal Care and Use Committee of Seoul National University (IACUC, SNU-200519–2), Korea. All procedures, including chicken maintenance, reproduction, and sample collection, were governed by standard operating protocols according to a standard management program at the University Animal Farm, Seoul National University and the Animal Genetic Engineering Laboratory at Seoul National University.

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