BLG gene knockout and hLF gene knock-in at BLG locus in goat by TALENs / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 329-338, 2016.
Article
in Chinese
| WPRIM
| ID: wpr-337411
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
Goats
/
Animals, Genetically Modified
/
Transfection
/
Chemistry
/
Milk
/
Nuclear Transfer Techniques
/
Gene Knock-In Techniques
/
Gene Knockout Techniques
/
Fibroblasts
Limits:
Animals
/
Female
/
Humans
/
Pregnancy
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2016
Type:
Article
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