Abstract
Nonrodent animal models have recently become more valuable in preclinical studies. The limitation of nonrodent animal models is that they must demonstrate relatively reliable and predictable responses in addition to representing complex etiologies of a genetically diverse patient population. In our study, we applied CRISPR/Cas9 technology to produce transgenic rabbits. This approach can be useful for creating genetically divergent and homogeneous populations for studies in translational medicine. NADPH oxidase 4 (NOX4) is a promising therapeutic target, as it is linked to several pathologies including stroke, atherosclerosis, and lung and kidney fibrosis. NOX4 knockout (KO) rabbit lines were created in order to study the in vivo effects resulting from a lack of NOX4 protein and loss of gene function. One of the knockout founders was a germline multiallelic knockout male. Its offspring segregated into three distinct NOX4 knockout and a wild-type lines. Mosaicism is a relatively frequent phenomenon in rabbit transgenesis. Our results point to the possible application of mosaicism in preclinical studies. However, careful planning and evaluation of results are necessary. The predicted off-target sites were studied as well, and no signs of off-target events were detected.
Highlights
Since the creation of the first genetic knockout rabbits with the CRISPR/Cas9 method in 2014 [1], the number of publications on CRISPR/Cas9-modified rabbits has increased, touching on a wide array of topics such as improving meat quality [2], altering fur color [3], studying the metabolic features [4,5], modeling human diseases [6], producing recombinant proteins [7], and improving the methodology [8,9,10]
The results of our study show the successful generation of several NADPH oxidase 4 knockout rabbit lines
The NADPH oxidase 4 (NOX4) gene plays an important role in biological processes, and reactive oxygen species have a modulatory effect on the physiological functions of diverse mammal organs
Summary
Since the creation of the first genetic knockout rabbits with the CRISPR/Cas method in 2014 [1], the number of publications on CRISPR/Cas9-modified rabbits has increased, touching on a wide array of topics such as improving meat quality [2], altering fur color [3], studying the metabolic features [4,5], modeling human diseases [6], producing recombinant proteins [7], and improving the methodology [8,9,10]. Because of their similar anatomy and bigger size, Appl. Sci. 2020, 10, 8508 experimentation is more achievable with rabbits, and they are considered to be a better subject for studies in translational medicine [18]. CRISPR/Cas technology in animal transgenesis has undergone great improvements in recent years. With the almost completely sequenced rabbit genome and expertise available, CRISPR/Cas is a relatively robust technology that can be used to knockout almost any gene in rabbits. Knocking out a gene with CRISPR/Cas is based on targeted cutting of double-stranded DNA and the cells’ own mechanism of DNA damage repair by nonhomologous end joining (NHEJ), resulting in indels at the target site that usually result an early stop codon that leads to fast degradation of mRNA [23,24]
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