Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer

Abstract

The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT+/− cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT+/− rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species.

Use of oocytes selected by brilliant cresyl blue staining enhances rabbit cloned embryo development in vitro

“…SCNT involves the injection of a somatic cell into a denucleated oocyte, followed by activation, culturing, and transfer to the oviduct or uterus, where the reconstructed embryo can develop into a new individual. SCNT allows gene editing to be performed in donor cells and allows the accuracy and safety of gene modification to be assessed before micromanipulation of the embryo and embryo transfer (Yin et al ., 2015). Consequently, SCNT is the main method that is used for the production of transgenic cattle, sheep, and pigs (Holm et al ., 2016; Tan et al ., 2016).…”

Rabbit Biomodels of Human Diseases Developed Using New Genomic Technologies. Crispr/Cas9 (Review)

“…Первый кролик с нокаутом гена (knockout, KO) был получен методом SCNT только в 2015 году [94]. Тем временем стремительно развивающиеся методы редактирования генома позволили получать KO-гены у разных видов животных, в т. ч. и кроликов [12,32].…”

The potential impact of new generation transgenic methods on creating rabbit models of cardiac diseases

“…Rabbit is model animal and -including the transgenic rabbits-it already has and will have its place among the different models of cardiac diseases. Contrary to the mouse, genetically modified embryonic stem cells or induced pluripotent stem cells do not contribute to germline transmission in rabbit, moreover to date the somatic cloning worked at very low efficiency in this species (Zakhartchenko et al, 2011), however recently the first knock out rabbit by nuclear transfer has been created (Yin et al, 2015).…”