Targeted gene knockout in chickens mediated by TALENs

Abstract

Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications.

Keywords: egg protein; genetic modification; germ-line chimera; poultry; programmable genome editing.

Fig. 1.

Genomic mutation analysis after TALEN introduction into chickens. (A) T7E1 assay of TALEN target sites for DsRed, B4GALT1, and β-actin in the chicken DF1 cell line. (B) Schematic TALEN design for the chicken OV gene. DNA-binding (left in red or right in blue) and spacer sequences (in black) are presented for OV TALEN 2. T7E1 assay of chicken DF1 cells mutated with OV TALEN 1 or 2 (Left), and chicken PGCs (SNUhp26) mutated with OV TALEN 2 (Right). (C) Mutated target DNA sequences of chicken PGCs with OV TALEN 2. A dash in the DNA sequences denotes the deleted nucleotides, and bold ATG indicates the translational initiation codon in the second exon of the OV gene.

Fig. 2.

(A) A germ-line chimeric male KOC founder (#4312) artificially inseminated a female WL. (B) The hatched chicks produced from the founder through test-cross and T7E1 assay of the offspring. Feather color of the donor WL (I/I)-derived chick was white, whereas the hybrid (I/i) chick produced from the recipient founder KOC had black spots. OV mutant chicks were identified by T7E1 assay. (C) Targeted DNA sequences in the OV gene were analyzed in the OV mutant donor PGC-derived offspring. A dash in the DNA sequences denotes deleted nucleotides, and bold ATG indicates the translational initiation codon in the second exon of the OV gene. (D) Detection of the OV TALEN 2 construct in mutant offspring by genomic PCR. OV TALEN 2 expression vector concentration was used as a series of 10-fold dilutions from 100 pg to 10 fg. Wild-type chicken genomic DNA was used as a negative control.