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Development of a Novel Gper‐1 Knock‐out Rat Model Using a Modified CRISPR/Cas9 Technology
Author(s) -
Waghulde Harshal,
Miller Shondra,
Filipaik Wanda,
Mell Blair,
Saunders Thomas,
Joe Bina
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.814.3
Subject(s) - gper , crispr , context (archaeology) , biology , cas9 , genetics , estrogen receptor , gene , microbiology and biotechnology , paleontology , cancer , breast cancer
The G‐protein coupled estrogen receptor ( Gper‐1 or Gpr 30 ) is a newly recognized estrogen receptor that is widely expressed in various tissues including heart and blood vessels. Rat Gper‐1 is a single exonic gene located on chromosome 12. Gper‐1 is implicated in the regulation of blood pressure in female mice potentially through its function as a receptor for estrogen. These studies conducted using mice that are without a genetic background permissive for the development of hypertension are not particularly useful for evaluating the function of Gper‐1 in the context of hypertension. To understand the function of Gper‐1 in the context of a genetically permissive background, we attempted to knock‐out the Gper‐1 gene on the genome of the Dahl‐salt sensitive (S) rat using an modified Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) method. To ensure complete knock‐out, instead of the traditional method of using a single gRNA, two gRNAs, each targeting one end of the 1128bp Gper‐1 gene were developed. The gRNAs were injected into the Dahl S rat embryos. The embryos were then implanted into ten pseudo‐pregnant females. Among the 125 pups born, 5 homozygous founders, 21 heterozygotes and 3 partial Gper‐1 deletion founders were identified. In conclusion, using an advanced CRISPR/Cas9 technique a panel of Gper‐1 rat knock‐outs and targeted mutants were generated, which will serve as novel models for studying the structure‐function relationships of the Gper‐1 gene. Research support to BJ: NHLBI/NIH 020176, 112641.