Generation Of β-Lactoglobulin Gene Knockout Goats By TALEN-Mediated Gene Targeting

β-Lactoglobulin(BLG) is a major goat’s milk allergen. Despite the use of biochemical approaches for reducing the allergenic potential of BLG, it could not radically solve the problem of BLG allergen. The development of targeted genome editing and transgenic animals can be applied for a more direct approach to reduce BLG levels in milk. It has been reported that transcription activator-like effector nucleases(TALENs)-mediated gene targeting enabled the precise genetic modification at the specifical genomic sites. In the present study, an effective method of using TALEN-mediated gene targeting in goat primary fibroblasts was described. TALENs and gene-targeting vectors against the goat BLG gene were constructed and co-transfected into goat fetal fibroblasts to introduce TALEN-mediated knockout of BLG gene. BLG mon-allelic targeted cells were used as donor for generation of BLG-targeted goats by nuclear transfer. After disruption of one allele, sequential gene targeting was applied to generate BLG bi-allelic knockout goats.1. Design and production of TALENs against the goat BLG geneExon 1 of the goat BLG gene was selected by the “TAL Effector-Nucleotide Targeter” as the target site for TALEN. DNA constructs of TALEN, p TSBE1-R and p TRBE1-L were produced by the “unit assembly” method. An RFP-GFP reporter system was used to determine the nuclease activities of p TSBE1-R and p TRBE1-L in 293 FT cells. The cells transfected with TALENs and their corresponding reporter construct yielded appreciable levels of red and green fluorescence, which indicated that these designed nucleases specifically cleaved the target site.2. Gene targeting vectors constructionTo disrupt the BLG locus, 3 knockout vectors were constructed. Both p BLG-neo and p BLG-neo-M contained a neomycin resistance(neo) gene driven by a phosphoglycerol kinase(PGK) promoter and the same 5’ homologous arm(1.1 kb). The 3’ arms were 5.3 kb and 1.3 kb for p BLG-neo and p BLG-neo-M, respectively. By contrast, the knockout vector p BLG-puro contained a EF1α-GFP-2A-puromycin cassette flanked by two 1.0 kb homologous arms.3. Targeting efficiency against the BLG gene in goat fetal fibroblastsTo introduce the frame-shift mutations that lead to the production of null alleles, TALENs’ DNA or m RNAs were delivered into goat fetal fibroblasts(GFFs) by electroporation to introduce NHEJ. Limited dilution was used to form cloned cells. No mutants were detected when a ~300 bp genomic fragment was amplified and sequenced from the 303 clones in any groups. To test whether the BLG gene could be disrupted by spontaneous HR, the targeting vector p BLG-neo and p BLG-neo-M were delivered into GFFs via electroporation, respectively. 1005 G418-resistant clones were selected, but none of them were correctly targeted. TALENs were introduced into GFFs with the targeting vectors and have significantly improved the targeting effecicency. Clones with the PGK-neo fragment site-specifically integration into BLG locous were detected by PCR analysis in all the groups. Meanwhile, we found that both 5? and 3? junction PCR analyses were necessary for screening gene targeting events. PCR analysis revealed that, 13.6%(n = 589) G418-resistant clones derived from GFFs transfected with TALENs-DNA constructs and p BLG-neo-M were correctly targeted; 10.7%(n = 459) G418-resistant clones derived from GFFs transfected with TALENs-econding m RANs and p BLG-neo-M were correctly targeted. Moreover, PCR analysis showed that the DNA fragment of TALEN vectors could be randomly integrated into the goat genome when TALEN was transfected into cells as DNA construct.4. Generation of BLG mono-allelic targeted goats by somatic cell nuclear transferTo eliminate the possible genomic integration of TALEN-DNA constructs, TALEN-encoding m RNAs-mediated targeted cells was used as donor nuclei for NT. 1128 cloned embryos were re-constructed and 7 cloned goats were born. PCR and Southern blot analysis revealed that the 7 goats were BLG mon-allelic targted. By sequencing the products of junction PCR, a 20 bp deletion of the BLG gene were found and a 1 935 bp selection gene were introduced into the cognate chromosomal location.5. Generating of BLG bi-allelic targeted goatsTo target the second allele of the BLG gene, BLG+/- fibroblasts were isolated from the ear tissues of mono-allelic targeted goats. TALEN-DNA constructs were introduced to form DSB at the intact allele in BLG+/- cells which produced bi-allelic targeting events via the secondary vector. PCR analysis revealed that, 5.85%(n = 667) puro-resistant clones derived from BLG+/- cells transfected with TALENs-DNA constructs and p BLG-puro were correctly targted; 5.10%(n = 647) puro-resistant clones derived from BLG+/- cells transfected with TALEN-encoding m RNAs and p BLG-puro were correctly targted. Southern blot analysis revealed that all the clones with positive PCR were bi-allelic targted in BLG locus. TALEN-encoding m RNAs-mediated BLG-/- cells were used as donor for NT. 805 cloned embryos were re-constructed and 3 cloned goats were born. PCR and Southern blot analysis showed that the 3 goats were BLG bi-allelic targeted.In conclusion, an effective approach for gene knockout using TALEN-mediated gene targeting in goat somatic cells was described. 7 BLG mon-allelic targeted goats and 3 bi-allelic targeted goats were generated by cloning which will be helpful for cultivation of new transgenetic biological varietie and for research of the BLG function.