Frontiers of Agricultural Science and Engineering
>> 2015,
Volume 2,
Issue 3
doi:
10.15302/J-FASE-2015059
RESEARCH ARTICLE
Generation of CRISPR/Cas9-mediated lactoferrin-targeted mice by pronuclear injection of plasmid pX330
1. State Key Laboratory of Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing 100193, China.2. College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
Accepted: 2015-07-06
Available online: 2015-11-10
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Abstract
Lactoferrin is a member of the transferrin family of multifunctional iron binding glycoproteins. While numerous physiological functions have been described for lactoferrin, the mechanisms underlying these functions are not clear. To further study the functions and mechanisms of lactoferrin, we modified the lactoferrin promoter of mice using the CRISPR/Cas9 system to reduce or eliminate lactoferrin expression. Seven mice with lactoferrin promoter mutations were obtained with an efficiency of 24% (7/29) by injecting the plasmid pX330, expressing a small guide RNA and human codon-optimized SpCas9, into fertilized eggs of mice. Plasmid integration and off-targeting of pX330 were not detected. These results confirmed that pronuclear injection of a circular plasmid is a feasible and efficient method for targeted mutagenesis in mice.
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