Abstract
Cytochrome P450 (CYP) 3A accounts for nearly 30% of the total CYP enzymes in the human liver and participates in the metabolism of over 50% of clinical drugs. Moreover, CYP3A plays an important role in chemical metabolism, toxicity, and carcinogenicity. New animal models are needed to investigate CYP3A functions, especially for drug metabolism. In this report, Cyp3a1/2 double knockout (KO) rats were generated by CRISPR-Cas9 technology, and then were characterized for viability and physiological status. The Cyp3a1/2 double KO rats were viable and fertile, and had no obvious physiological abnormities. Compared with the wild-type (WT) rat, Cyp3a1/2 expression was completely absent in the liver of the KO rat. In vitro and in vivo metabolic studies of the CYP3A1/2 substrates indicated that CYP3A1/2 was functionally inactive in double KO rats. The Cyp3a1/2 double KO rat model was successfully generated and characterized. The Cyp3a1/2 KO rats are a novel rodent animal model that will be a powerful tool for the study of the physiological and pharmacological roles of CYP3A, especially in drug and chemical metabolism in vivo.
Highlights
The Cytochrome P450 (CYP) enzymes play an essential role in the biotransformation of endogenous molecules and xenobiotics[1]
For targeting Cyp3a1, we selected 5′-CAAGAAACAGGGGATTCC-3′followed by TGG as the target site, and 5′-TAAGAAACAAGGAATTCC-3′followed by TGG for targeting Cyp3a2
To identify the gene modifications of the F0 generation, the targeted loci of Cyp3a1 and Cyp3a2 were PCR amplified and T7E I (T7 endonuclease I) cleavages were detected in rat #3, #5, #6, #8, #11 and #12 founders for Cyp3a1 and in #3, #5, #7, #9, #12 and #13 founders for Cyp3a2 (Fig. 2a), which indicated the potential for genome modification at targeted loci
Summary
The Cytochrome P450 (CYP) enzymes play an essential role in the biotransformation of endogenous molecules and xenobiotics[1]. A variety of single CYP enzyme-null mouse models, including Cyp3a, have been reported as potent and precise tools used to illustrate the potential functions of CYP isoforms on the metabolism, toxicity and carcinogenicity of chemicals[6,7]. Compared with the Cyp3a KO mouse model, Cyp3a KO rat model is more important to pharmacological research, especially drug metabolism and pharmacokinetic (DMPK) studies. Since many CYP isoforms expressed in different species possess different substrate affinities, it is very difficult to extrapolate the results from one specific animal species to humans[4]. To take advantages of rats in DMPK and disease research and to enrich resources of animal model in pharmacology, we want to generate a Cyp3a1 and 3a2 double KO rat model via the CRISPR-Cas[9] system. The Cyp3a1/2 double KO rat was viable, fertile, physiological normal and presented impaired metabolic ability towards selected CYP3A probe substrates
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