Non-equivalence of cloned and clonal mice

Cloning mice by nuclear transfer is conventionally performed using a one-step procedure. Recently an alternative two-step procedure has been proposed for generating mice that are clonally derived from a single somatic donor nucleus. Here, we demonstrate that such mice may not be clones of the original donor nucleus. In the conventional one-step cloning by nuclear transfer (nt), a donor cell nucleus is transferred into an enucleated mouse oocyte. Following activation, the resulting embryo is cultured in vitro and transferred to the uterus of a recipient [1]. In the alternative two-step procedure [2,3], a cloned blastocyst is used in the first step to derive a nuclear transfer-embryonic stem cell (ntES) cell line in vitro. In the second step, multiple ntES cells of a given line are injected into a tetraploid blastocyst that was derived from a fertilized oocyte. ES cells, and by inference ntES cells, do not contribute well to extra-embryonic tissues, but tetraploid cells can complement this deficiency. Conversely, tetraploid cells have a competitive disadvantage in colonizing the embryo proper. In principle, however, injection of ntES cells into tetraploid blastocysts results in chimeric mice [4] unless proven otherwise. Nonetheless, mice generated from B and T lymphocytes or olfactory sensory neurons by this two-step procedure have been referred to as 'cloned' [2,3], based on the premises that all of the cells in their bodies are descendants of the injected ntES cells, and that the nuclear genome of these cells is an exact copy of that of the original donor cell. Here, we have tested these premises. We and others have proposed to refer to mice produced by this two-step method as 'clonal' [5,6]. We established the ntES cell line M71CZ3 by nuclear transfer from a genetically marked, GFP-positive olfactory sensory neuron expressing the M71 odorant receptor gene [5]. The neuron came from a male mouse that was heterozygous for the M71-IRES-Cre targeted mutation [5]. We injected M71CZ3 cells into tetraploid blastocysts generated by electrofusion of diploid embryos from wild-type B6D2F1 females mated with hemizygous Z/EG males. The Z/EG transgene provides widespread and constitutive expression of β β-galactosidase [7], which serves as a marker for tetraploid cells in our design. (The GFP gene and Cre-mediated recombination between loxP sites are not relevant for our purpose here.) In 619 tetraploid complementations, 39 clonal mice developed to term (6.3%). They were sacrificed soon after birth. The placentae of 11 clonal newborns showed intense X-gal …