title: [BLG gene knockout and hLF gene knock-in at BLG locus in goat by TALENs].
abstract: To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
authors.forename: Shaozheng
authors.initials: S
authors.lastname: Song
authors.forename: Mengmin
authors.initials: M
authors.lastname: Zhu
authors.forename: Yuguo
authors.initials: Y
authors.lastname: Yuan
authors.forename: Yao
authors.initials: Y
authors.lastname: Rong
authors.forename: Sheng
authors.initials: S
authors.lastname: Xu
authors.forename: Si
authors.initials: S
authors.lastname: Chen
authors.forename: Junyan
authors.initials: J
authors.lastname: Mei
authors.forename: Yong
authors.initials: Y
authors.lastname: Cheng
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D005260:Female; D005347:Fibroblasts; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D006041:Goats / Q000235:genetics*; D006801:Humans; D007781:Lactoferrin / Q000235:genetics*; D007782:Lactoglobulins / Q000235:genetics*; D008892:Milk / Q000737:chemistry; D053652:Nuclear Transfer Techniques; D010957:Plasmids; D011247:Pregnancy; D014162:Transfection
chemical_list: C490098:LTF protein, human; D007782:Lactoglobulins; D007781:Lactoferrin