Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation

Abstract

To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F(1) genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F(1) ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F(1) ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F(1) ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F(1) ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F(1) ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate.

Figure 1

In vitro preimplantation development of ES cell clones. Values are displayed as the percentage of embryos reaching each developmental stage. For PN formation rate, efficiency is expressed as the percent of total oocytes surviving reconstruction for each ES cell line. In the case of two-cell and blastocyst stage development, efficiency is expressed as the percent of embryos of all with PN. Data from two independent 129/Sv × 129/Sv ES cell lines were similar and therefore combined.

Figure 2

Hemotoxylin and eosin staining of lung sections from ES cell-derived and control neonatal mice. Neonatal mice delivered by cesarean section were observed for 45 min for respiration. After observation, lungs were removed and placed overnight in Bouin's fixative. After fixation, tissue was paraffin-embedded, sectioned, and stained. Magnification: ×40.

Figure 3

Birth (A) and placental (B) weights of ES cell-derived and control neonatal mice. Birth and placental weights of clones were significantly higher than either ES cell-tetraploid mice (P < 0.0001 for both weights) or in vitro-cultured controls (P < 0.0001 for both weights) in pair-wise comparisons. Neither birth nor placental weights of ES cell-tetraploid mice were significantly different from in vitro-cultured controls (P > 0.05 for both weights). Birth and placenta weights of normal mice were significantly lower than cloned, ES cell-tetraploid or in vitro-cultured pups (for all P < 0.004). Pairwise comparisons were performed by using the Student's t test. Data from normal pups were recorded from litters with a size less than or equal to three. Cross-bars mark the mean weight for each data set. In vitro-cultured, control animals were generated by isolating two-cell stage embryos, culturing them to the blastocyst stage, and then transferring them to recipient females.

Figure 4

ES cell clones display increased neonatal birth and placental weight. These two animals were derived from the same ES cell line, F_1.2–3, one cloned by nuclear transfer, the other derived by tetraploid embryo complementation. Note the dramatic increase in both neonatal and placental size in the cloned pup. (Bars = 1 cm.)