The Derivation of Highly Germline-Competent Embryonic Stem Cells Containing NOD-Derived Genome

Abstract

It would be extremely advantageous to the analysis of disease mechanisms in the spontaneous mouse model of type 1 diabetes, the nonobese diabetic (NOD) strain, if genes in this strain could be modified in vivo using embryonic stem (ES) cells and homologous recombination. However, a NOD ES cell line with adequate germline transmission has not yet been reported. We report the development of highly germline-competent ES cell lines from the F1 hybrid of NOD and 129 for use in NOD gene targeting. Consequently, we developed ES cell lines derived from (NOD ؋ 129)F1 ؋ 129 backcross 1 mice, which were intercrossed to select for homozygosity of particular regions of NOD genome known to contain disease loci. Diabetes 52: [205][206][207][208] 2003 T he nonobese diabetic (NOD) mouse is a valuable model of human type 1 diabetes, particularly since the most important gene, the immune reponse gene IAb, and its human orthologue DQB1 share at least one of the same causal variants. However, allelic variation of this gene is not sufficient to explain disease and other genes are involved. Identification and functional analysis of candidate genes would be greatly facilitated if they could be altered in vivo by homologous recombination. Unfortunately, derivation of embryonic stem (ES) cells from the NOD mouse has proved extremely difficult. In addition, the modification of genes in ES cells from other strains such as 129 can lead to ambiguous results due to the unpredictable influence of linked 129-derived genes on the development of diabetes following the backcrossing of the chimeras to the NOD background. Thus far there is only one report of an NOD ES cell line that has demonstrated germline competence (1), but both its rate of growth and the level of germline transmission are too low to enable its use in gene targeting. We have had previous success in deriving ES cell lines from refractory strains of mice using microsurgery to explant epiblast of blastocysts that have been subjected to implantation delay (2). Here we present the results of using these same techniques, first with the NOD mouse and then with (NOD ϫ 129)F1 hybrid embryos.Our initial attempts to derive NOD ES cells involved ovariectomizing NOD mice on the third day of pregnancy to initiate implantation delay, and recovering the delayed blastocysts 7 days later. However, the number of blastocysts recovered was extremely low. Of 22 pregnant females, 18 yielded none and the remaining 4 gave only 17 living blastocysts. In contrast, of 38 nonovariectomized NOD females, 36 were pregnant with an average of nine blastocysts. This suggests that blastocyst viability is severely compromised by delaying implantation in the NOD mouse.In subsequent experiments, NOD conceptuses were explanted on the first, third, or fourth day of pregnancy and transferred to non-NOD host dams in which implantation delay was then induced by ovariectomy. Epiblasts from a total of 372 delayed blastocysts were placed in culture, and 9 of these gave rise to colonies of cells that had a typical ES cell ...

Differentiation and Transplantation of Functional Pancreatic Beta Cells Generated from Induced Pluripotent Stem Cells Derived from a Type 1 Diabetes Mouse Model

“…The NOD mouse strain is considered a nonpermissive strain for ESC derivation [48], because ESC derivation requires specific exogenous factors to maintain the ICM-like pluripotent state [44]. In our study, when the magnet-based nanofection technique was used for generation of exogenous DNA-free iPS cells, we could not stably establish the NOD-iPSCs.…”

Development of single mouse blastomeres into blastocysts, outgrowths and the establishment of embryonic stem cells

“…Although there have been several attempts to establish mouse ES cell lines from CD-1 stock (Suzuki et al 1999, Brook et al 2003, reports on the derivation of CD-1 ES cell lines are very limited. Wakayama et al (2005) have attempted to use somatic cells from male and female CD-1 mice as donor cells for nuclear transfer (NT) and for the establishment of ES cells from the reconstructed blastocyst (Wakayama et al 2005).…”

Derivation of germ-line-competent embryonic stem cell lines from preblastocyst mouse embryos

“…These denuded embryos were either explanted individually into separate 1.9-cm 2 wells that had been seeded 24-72 h earlier with PEFs irradiated with 30 gray (feeders) or into a microdrop of Ca 2ϩ , Mg 2ϩ -free PBS in 1.9-cm 2 wells. Feeders were plated at a density of 7.5 ϫ 10 4 cells per cm 2 . Embryos that were deliberately ''stuck'' to the dish in the PBS microdrops were carefully flooded with a feeder cell suspension after an interval of 5 min.…”

“…Previous work established that ES cells derived from the late inner cell mass (ICM) originate exclusively from the epiblast rather than the primitive endoderm or trophectoderm (1). Although starting with epiblast isolated from advanced blastocysts greatly increased the ease of obtaining ES cells, certain strains such as ICR and the NOD strain derived therefrom still proved to be largely refractory with this method (2). The possibility exists that mouse strains may differ in the period of development that readily yields ES cell lines.…”