Supporting Information

Transcription

1 Supporting Information Park et al /pnas TCAAGTCCATCTACATGGCC-3 5 -CAGCTGCCCGGCTACTACTA-3 5 -TGCAGCTGCCCGGCTACTAC-3 5 -AAGCTGGACATCACCTCCCA-3 5 -TGACAGGAACACCTACAAGT-3 5 -AAGGCACCTTTCTGTCTCCA-3 5 -TTAGTGATGTAGACCTGATC-3 5 -AGGAACACCTACAAGTGCTA-3 5 -TGAGAGTAGCCCCTGAGGAG-3 5 -CTCACAGAGGCTCCCCTGAA-3 5 -TGTGCTGCTCACAGAGGCTC-3 5 -AGCCAACAGAGAGAAGATGA-3 Fig. S1. Transcription activator-like effector nuclease (TALEN) target sites and spacer lengths in chicken genome target genes (DsRed, β1,4-galactosyltransferase 1, and β-actin). 1of5

2 TAGTAGTAGCCGGGCAGCTGCACGGGCTTCTTGGCCATGTAGATAGACTTGA wild (4X) TAGTAGTAGCCGGGCAGCTGCACGGcCTTCTTGGCCATGTAGATAGACTTGA 1nt indel (1x) TAGTAGTAGC TAGACTTGA 33nt del (1x) TAGTAGTAGCCGGGCAGCTGCACG---TTCTTGGCCATGTAGATAGACTTGA 3nt del (1x) TAGTAGTAGCCGGGCAGCTGCACGG ACTTGA 21nt del (1x) TAGTAGTAGCCGGG ATAGACTTGA 28nt del (1x) TAGTAGTAGCCGGGCAGCTGCACGGG CATGTAGATAGACTTGA 9nt del (1x) TGGGAGGTGATGTCCAGCTTGGCGTCCACGTAGTAGTAGCCGGGCAGCTGCA wild (5x) TGGGAGGTGATGTCCAGCTTGGCGTC------GTAGTAGCCGGGCAGCTGCA 6nt del (1x) TGGGAGGTGATGTCCAGCTTGGCGT AGCCGGGCAGCTGCA 12nt del (1x) TGGGAGGTGATGTCCAGCTTGGCGT---aGTAGTAGTAGCCGGGCAGCTGCA 4nt indel (1x) TGGGAGGTGATGTCCAGCT------CCACGTAGTAGTAGCCGGGCAGCTGCA 6nt del (1x) TGGGAGGTGATGTCCAGCTTGGCGTCCA---AGTAGTAGCCGGGCAGCTGCA 3nt del (1x) TGGGAGGTG TAGCCGGGCAGCTGCA 27nt del (1x) TGACAGGAACACCTACAAGTGCTACAGCCAACCAAGGCACCTTTCTGTCTCCA wild (4x) TGACAGGAACACCTACAAGTGCTA--GCCAACCAAGGCACCTTTCTGTCTCCA 2nt del (1x) TGACAGGAACACCTACAAGTGCTAC---CAACCAAGGCACCTTTCTGTCTCCA 3nt del (1x) TGACAGGAACACCTACAAGTGCTAC CAAGGCACCTTTCTGTCTCCA 7nt del (1x) TGACAGG GCACCTTTCTGTCTCCA 29nt del (1x) TTAGTGATGTAGACCTGATCCCAATGGATGACAGGAACACCTACAAGTGCTA wild (4X) TTAGTGATGTAGACCTGATCCCAA CAGGAACACCTACAAGTGCTA 7nt del (1x) TTAGTGATGTAGACCTGATCCCAA--GATGACAGGAACACCTACAAGTGCTA 2nt del (2x) TTAGTGATGTAGACCTGATCCCAATGG-TGACAGGAACACCTACAAGTGCTA 1nt del (1x) TTAGTGATGTAGACCTGATCC AGGAACACCTACAAGTGCTA 11nt del (1x) TTAGTGATGTAGACCTGATCCCA GGAACACCTACAAGTGCTA 10nt del (1x) TTCAGGGGAGCCTCTGTGAGCAGCACAGGGTGCTCCTCAGGGGCTACTCTCA wild (10x) TTCAGGGGAGCCTCTGTGAGCAGC-----GTGCTCCTCAGGGGCTACTCTCA 5nt del (1x) β-actin TALEN# TCATCTTCTCTCTGTTGGCTTTGGGGTTCAGGGGAGCCTCTGTGAGCAGCACA wild (11x) TCATCTTCTCTCTGTTGGCTTTGGGGT--AGGGGAGCCTCTGTGAGCAGCACA 2nt del (1x) Fig. S2. DF1 mutation analysis after transfection of each TALEN construct and FACS. The target loci were analyzed by DNA sequencing. (A) For the DsRed reporter gene, the negative cell population was separated for sequencing analysis. (B) β1,4-galactosyltransferase 1 gene and (C) β-actin gene. For both target genes, GFP-positive cells were isolated and analyzed. A dash in the DNA sequences denotes deleted nucleotides; inserted nucleotides are indicated by a lowercase letter. 2of5

3 TGCTCTAGACAACTCAGAGTTCACCATGGGCTCCATCGGTGCAGCAAGCATGGA 5 -TCCATGCTTGCTGCACCGAT-3 5 -TGCTCTAGACAACTCAGAGT-3 TGCTGTTTGCTCTAGACAACTCAGAGTTCACCATGGGCTCCATCGGTGCAGCAAGCA 5 -TGCTTGCTGCACCGATGGAG-3 5 -TGCTGTTTGCTCTAGACAAC-3 M G S I G A A S amino acids TGCTGTTTGCTCTAGACAACTCAGAGTTCACC ATG GGC TCC ATC GGT GCA GCA AGC A wild (19x) TGCTGTTTGCTCTAGACAACTCAGAG GGC TCC ATC GGT GCA GCA AGC A 9nt del (2x) TGCTGTTTGCTCTAGACAACTCAGAG ATG GGC TCC ATC GGT GCA GCA AGC A 6nt del (2x) TGCTGTTTGCTCTAGACAACTCAGAGTTCA GGC TCC ATC GGT GCA GCA AGC A 5nt del (1x) TGCTGTTTGCTCTAGACAACTCAGAGT----C ATG GGC TCC ATC GGT GCA GCA AGC A 4nt del (1x) TGCTGTTTGCTCTAGACAACTCAGAGT-CACC ATG GGC TCC ATC GGT GCA GCA AGC A 1nt del (1x) TGCTGTTTGCTCTAGACAACTCAGAGTTcgttCACC ATG GGC TCC ATC GGT GCA GCA AGC A 4nt in (3x) TGCTGTTTGCTCTAGACAACTCAGAGTTcatcCACC ATG GGC TCC ATC GGT GCA GCA AGC A 4nt in (1x) Fig. S3. (A) Diagram of ovalbumin (OV) TALEN target sites in the chicken OV gene. DNA binding (left in red or right in blue) and spacer sequences (black) are presented for OV TALENs. (B) Mutated target DNA sequences in DF1 cells with OV TALEN 2. A dash in the DNA sequences denotes deleted nucleotides; inserted nucleotides are represented by a lowercase letter. Bold ATG indicates the translational initiation codon in the second exon of the OV gene. Fig. S4. (A) Male SNUhp26 primordial germ cell (PGC) line. (B) Detection of GFP-expressing PGCs before and after FACS separation under a fluorescence microscope. (C) Separation of GFP-positive PGCs 1 d after lipofection with the CMV-GFP construct and OV TALEN 2 by FACS. 3of5

4 M G S I G A A S amino acids TGCTGTTTGCTCTAGACAACTCAGAGTTCACC ATG GGC TCC ATC GGT GCA GCA AGC A wild (13x) TGCTGTTTGCTCTAGACAACTCAGAGT CA GCA AGC A 21nt del (2x) TGCTGTTTGCTCTAGACAACTCAGAGTTC TC GGT GCA GCA AGC A 13nt del (1x) Fig. S5. (A) In founder KOCs, donor WL-derived sperm was detected with strain-specific primers. All founders produced donor PGC-derived sperm as well as the recipient KOC sperm. (B) T7 endonuclease 1 assay in male founder sperm that were transplanted into the OV TALEN 2-mutated male PGCs. (C) Mutant genotypes were analyzed in founder #4312 sperm. The sperm had a genotype similar to that of mutant PGCs. TGCTGTTTGCTCTAGACAACtcagagttcaccatgggCTCCATCGGTGCAGCAAGCA TGCTcTTTaCTCcAGAaAACagctttctaacagctcctCTCCATatGTGCtGtAAtCA TGCTaTTacCTCaAcACAACactgcttaaatagcttattaTCgATtGGTtCAcgAAGCA TGCTcTTTcCTgTAGcCAACatggggttactgctaccaggttctaacctTgCtTtGtTGagGCAAGCA Fig. S6. (A) Potential off-target analysis of OV TALEN 2 in mutant PGCs and offspring (#648 and #704). No off-target mutants were observed in offspring or mutant PGCs. (B) The experimental strategy for generating the targeted gene knockout chickens mediated by TALENs. WLs with a dominant pigmentation inhibitor gene (I/I) and KOCs with a recessive pigmentation inhibitor gene (i/i) were used for the donor PGCs and the recipient embryos, respectively. Through test-cross analysis by mating with WL hens (I/I), the germ-line chimeras were identified by offspring phenotype. Finally, the progenies derived from the transplanted mutant PGCs were screened using T7 endonuclease 1 assay, and the mutant genotype of individual chicks was subsequently identified by DNA sequencing. 4of5

5 Table S1. Number of injected recipients Hatchability of the recipient founders after injection of ovalbumin-mutated PGCs Number of hatched chicks Number of male chickens Number of sexually mature chickens (30.0%) 15 (55.6%) 10 (66.7%) Table S2. Potential target-off sites of chicken ovalbumin TALEN 2 Chromosome locus Direction Sequences* Ovalbumin (chromosome ) TGCTGTTTGCTCTAGACAACtcagagttcaccatgggCTCCATCGGTGCAGCAAGCA Chromosome TGCTcTTTaCTCcAGAaAACagctttctaacagctcctCTCCATatGTGCtGtAAtCA Chromosome TGCTaTTacCTCaAcACAACactgcttaaatagcttattaTCgATtGGTtCAcgAAGCA Chromosome TGCTcTTTcCTgTAGcCAACatggggttactgctaccaggttctaacctTgCtTtGtTGagGCAAGCA *Putative target binding sequences are in bold and identical target nucleotides are in uppercase. Spacer sequences are underlined. Table S3. Primer sequences and PCR conditions for amplification of DsRed, β1,4-galactosyltransferase 1, β-actin, and ovalbumin genes Gene and primer sequences PCR product, bp Annealing temperature, C DsRed Forward: 5 -GGCAAACAACAGATGGCTGG Reverse: 5 -CCGACATCCCCGACTACAAG β1,4-galactosyltransferase 1 Forward: 5 -GCTTTCCAGGCTAGGCAGAA Reverse: 5 -TGACCATGTGGCTGCTCTAC β-actin Forward: 5 -ACGGTATAGTCACTCAAAGATGGT Reverse: 5 -GTGTGGCACCTGGAAAGAAAAC Ovalbumin Forward: 5 -CTGGGACAGTTTGCTACCCA Reverse: 5 -CTGGTGCTGTCTTTTGCACC Ovalbumin (nested) Forward: 5 -ATAGAGTACCCTGCATGGTA Reverse: 5 -CAGGTATACCATGGCTAGAG 5of5