Propagation of an infertile hermaphrodite mouse lacking germ cells by using nuclear transfer and embryonic stem cell technology

Abstract

Animals generated by systematic mutagenesis and routine breeding are often infertile because they lack germ cells, and maintenance of such lines of animals has been impossible. We found a hermaphrodite infertile mouse in our colony, a genetic male with an abnormal Y chromosome lacking developing germ cells. We tried to clone this mouse by conventional nuclear transfer but without success. ES cells produced from blastocysts, which had been cloned by using somatic cell nuclear transfer (ntES cells) from this mouse, were also unable to produce offspring when injected into enucleated oocytes. Although we were able to produce two chimeric offspring using these ntES cells by tetraploid complementation, they were infertile, because they also lacked developing germ cells. However, when such ntES cells were injected into normal diploid blastocysts, many chimeric offspring were produced. One such male offspring transmitted hermaphrodite mouse genes to fertile daughters via X chromosome-bearing sperm. Thus, ntES cells were used to propagate offspring from infertile mice lacking germ cells.

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). A much lower morula/blastocyst rate resulted when compared with B6D2 mice.…”

Successful Mouse Cloning of an Outbred Strain by Trichostatin A Treatment after Somatic Nuclear Transfer

“…he success of somatic cell cloning in the mouse brings promise to applications such as species preservation, livestock propagation, and cell therapy for medical treatment using nuclear transfer embryonic stem cells (NT-ES cells) [1][2][3][4]. However, mouse cloning by somatic cell nuclear transfer (SCNT) has been inefficient, with success rates of 2% or less since the first cloned mouse, "Cumulina", was born in 1997 [5].…”

Improvement of mouse cloning using nuclear transfer-derived embryonic stem cells and/or histone deacetylase inhibitor

“…The cloned mice were obtained from these ntES cells by a second round of NT . We also achieved success using a mutant, hermaphroditic, sterile mouse found in our ICR mouse breeding colony (Wakayama et al, 2005a). Although the mutant mouse died accidentally soon after tail-tip biopsy and we failed to produce cloned mice from those cells, we could establish several ntES cell lines.…”

“…These can be used as an unlimited source of donor nuclei for NT and can therefore complement conventional somatic cell NT cloning approaches (Wakayama et al, 2005b). Unfortunately, if difficulty is experienced in producing cloned mice from a donor cell, it is also experienced when attempting to produce cloned mice by a second round of NT from ntES cell nuclei (Wakayama et al, 2005a).…”

“…Even in infertile and 'unclonable' strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology Wakayama et al, 2005a;Wakayama et al, 2005b). Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or cells Wakayama et al, 2008), such as from frozen extinct animals.…”