Hybrid Embryonic Stem Cell-Derived Tetraploid Mice Show Apparently Normal Morphological, Physiological, and Neurological Characteristics

Abstract

ES cell-tetraploid (ES) mice are completely derived from embryonic stem cells and can be obtained at high efficiency upon injection of hybrid ES cells into tetraploid blastocysts. This method allows the immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps. To provide a baseline for the analysis of ES mouse mutants, we performed a phenotypic characterization of wild-type B6129S6F 1 ES mice in relation to controls of the same age, sex, and genotype raised from normal matings. The comparison of 90 morphological, physiological, and behavioral parameters revealed elevated body weight and hematocrit as the only major difference of ES mice, which exhibited an otherwise normal phenotype. We further demonstrate that ES mouse mutants can be produced from mutant hybrid ES cells and analyzed within a period of only 4 months. Thus, ES mouse technology is a valid research tool for rapidly elucidating gene function in vivo.The standard protocol to derive mouse mutants currently requires the production of germ line chimeras from heterozygous targeted embryonic stem (ES) cells, followed by at least two breeding steps to obtain homozygous mutants (2). Thus, the production of a mutant strain is a time-intensive task exceeding 12 months prior to the analysis of adult mutants. In addition, substantial resources are required for the breeding and genotyping of several hundred mice involved in a typical knockout project. Besides this classical approach, conditional gene targeting through Cre/LoxP-mediated recombination is increasingly used as it allows the spatial and temporal control of gene inactivation (13,21). Given that a Cre transgene needs to be introduced via additional breeding steps, the production of conditional knockout mice involves four reproductive cycles requiring at least 16 months before the target gene's function can be analyzed in vivo.Due to these extensive timelines, the impact of targeted mutants in high-throughput functional genome analysis is currently limited, creating a demand for a time-saving single-step procedure. Cloning of mice does not provide a viable alternative because the nuclear transplantation procedure is inefficient and a variety of abnormalities have been described in cloned mice which likely result from incomplete genome reprogramming (8,17,25,30). Alternatively, ES cell-tetraploid (ES) mice can be produced in a single step through the introduction of diploid ES cells into tetraploid blastocysts (16). The latter provide an initial host environment for the differentiation of ES cells but do not contribute to the embryo at later developmental stages. Although the methodology to produce ES mice from inbred ES cell lines was described more than a decade ago, its application was limited due to the extremely low frequency at which viable ES pups are recovered (16).Recently, this technology was significantly improved through the discovery that ES cell lines de...

Living with or without cyclins and cyclin-dependent kinases

“…Rescue of this defect in extraembryonic tissues by tetraploid blastocyst complementation yielded animals that survived to term, with cardiac anomalies of varying severity but otherwise normal development . (Although this technique guarantees that ES cells contribute only to the embryo proper, mice die of respiratory failure after birth, a process that can be bypassed by the use of hybrid ES cell strains [Schwenk et al 2003]. Therefore, postnatal cyclin E1-and E2-deficient mice derived from an inbred ES cell strain did not survive.)…”

The genetic heterozygosity and fitness of tetraploid embryos and embryonic stem cells are crucial parameters influencing survival of mice derived from embryonic stem cells by tetraploid embryo aggregation

“…Pluripotent ES cells have been used extensively in studies of embryogenesis, gene function and development in the mouse. Tetraploid mouse embryos are not capable of completing normal development independently (Kaufman & Webb 1990, Eakin & Behringer 2003, but when they are complemented by the introduction of ES cells, they can then develop into normal conceptuses derived completely from ES cells (Ueda et al 1995, Nagy et al 1990, 1993, Wang et al 1997, Eggan et al 2001, Schwenk et al 2003, Wang & Jaenisch 2004.…”

“…With development some adult ES mice were generated from R1 ES cells derived from an intercross between two different 129 substrains (129/Sv and 129/Sv-CP; Nagy et al 1993). The genetic heterozygosity of ES cells is a crucial parameter influencing postnatal survival of ES mice and adult ES mice can be generated more successfully from hybrid ES cells in culture or after consecutive rounds of drug selection (Eggan et al 2001, Schwenk et al 2003. In contrast to cloned mice, ES mice have smaller birth and placental weights (Eggan et al 2001) and show apparently normal morphological, physiological and neurological characteristics (Schwenk et al 2003).…”

Methods to Generate Chimeric Mice from Embryonic Stem Cells

“…The second popular method is microinjecting ES cells into 4n blastocysts (Eggan et al, 2001;Kirchain et al, 2008;Li et al, 2005;Lin et al, 2010;Schwenk et al, 2003;Wang et al, 1997;Wang & Jaenisch, 2004). Most tetraploid-ESC neonates derived from hybrid ES cells developed into fertile adults (Eggan et al, 2001).…”

“…Previous studies also revealed that an ESC-derived mouse have host embryo contamination (Eggan & Jaenisch, 2003;Li et al, 2005), poor viability, and other minor abnormalities such as altered growth rate and body weight. However, adults had normal morphological, physiological, and neurological characteristics (Schwenk et al, 2003). Using 4n complementation method, 4n embryos require produced of 2n 2-cell embryos usually by electrofusion and are less viable than normal 2n embryos.…”