Somatic cell cloning involves replacing the nucleus of an oocyte with that of a somatic cell, which then directs development of the reconstructed embryo into a new organism with a nuclear genome identical to that of the donor cell1. Cloning using somatic cells cultured long-term2 laid the foundation for single-gene targeting in species other than mouse3. But targeting multiple genes by cloning has not been possible because of the limited lifespan of primary somatic cells and the long gestational times of large animals. The paper by Yoshimi Kuroiwa and colleagues4 on page 775 reports a sequential targeting technique that integrates homologous recombination, positive and negative selection, and Cre-loxP recombination with serial cloning to rejuvenate somatic cells and facilitate modification of multiple genes in a single cloned animal (Fig. 1a).

Figure 1: Rapid generation of homozygous double-knockout livestock.
figure 1

(a) Transfection of fibroblasts with a targeted gene knockout (KO) construct, followed by nuclear transfer, cloning to rejuvenate new primary fibroblasts and subsequent rounds of transfection and cloning, yields double-knockout cells from which a cloned cow can be derived in less than 2 years. (b) Generating homozygous single-gene knockout (KO) cows by conventional means would take ∼5 years from the birth of the first cloned cows.

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Rejuvenation by serial cloning

Generating a homozygous single-gene knockout cow would take ∼6 years using conventional methods (Fig. 1b). First, a gene of interest would be knocked out by homologous recombination in cultured somatic cells (typically fibroblasts), which would then be cloned to create a heterozygous calf with a targeted mutation. The heterozygotes would be raised to maturity for breeding (∼2 years of age) to generate first-generation (F1) male and female heterozygous calves, which would be cross-bred to generate a homozygous knockout calf. Generating homozygotes with respect to multiple targeted mutations using the conventional approach in cattle would be impractical.

In contrast, the sequential targeting strategy described by Kuroiwa et al. makes multiple genetic modifications in cattle, allowing the generation of homozygous cows in less than 2 years (Fig. 1a). First, the targeted cloned embryos are transferred to foster mothers to generate fetuses (not for term development, but for rejuvenating somatic cells for the next round of gene targeting). The rejuvenated fetal cells are then collected and subjected to a second round of homologous recombination to target the second allele, thereby generating homozygous knockout fetuses. Using this approach, each targeting event requires only 2.5 months, and a calf homozygous with respect to two targeted genes can be produced in less than 24 months (Fig. 1a).

The third and fourth rounds of marker selections present a challenge for sequentially targeting a second gene. To circumvent this problem, the authors recycled the same selectable markers5, neor and puror, in their sequential gene targeting efforts, using the Cre-loxP system to remove the drug resistance markers used in the first and second rounds of gene targeting. This allows them to use limited selection markers to target multiple genes in a single animal. The neor and puror marker genes were not expressed in the cloned fetuses, probably owing to methylation of these genes in the donor cells during cloning reprogramming, as transgenes are often silenced by DNA methylation in transgenic animals6. Whether silencing of the marker genes will be a persistent feature of cloning or a random inactivation phenomenon has yet to be determined. The finding that the Cre recombinase plasmid integrated into the genome in most fetuses was also unexpected, but this problem might be solved with further technical improvements to the methodology.

Primary somatic cells have a finite lifespan in culture and would probably become senescent after one or two rounds of gene targeting. Kuroiwa et al. used serial cloning to rejuvenate primary somatic cells in order to accomplish multiple rounds of gene targeting. Cell rejuvenation in clones by rebuilding of eroded telomeres from aged donor cells in cattle7,8 has been confirmed previously. Telomere reprogramming has also been found after serial cloning in cattle9 and mice10. Notably, the pregnancy success rate after four rounds of fetal serial cloning was as high as 71%; however, the efficiency for term development is not yet known. The discrepancy between this study and previous serial cloning reports9,10, which found progressive decreases in cloning efficiency, is probably due to the different source of donor cells (adult versus fetal cells).

Bigger, better, faster

Sequential targeting will undoubtedly be used to produce valuable livestock and create cattle resistant to deadly diseases, such as bovine spongiform encephalopathy (BSE). BSE is believed to be caused by prions, aberrant forms of a normal prion protein, PrP. Approximately 200,000 cattle have been diagnosed with BSE11 and more than 130 people have died from BSE12 since the first outbreak in 1996. This and previous studies11,12 indicate that PRNP may be knocked out to create cows resistant to BSE. Although certain lines of PrP knockout mice are healthy, the health of PrP knockout cattle and the effectiveness of making such cattle resistant to BSE infection have not yet been tested.

Another benefit of mutating PRNP applies to using cattle as bioreactors. Medical products such as human immunoglobulin and human serum albumin produced in transgenic cattle must be quality-controlled to be prion-free. Mutating PRNP in these cattle could help ensure that bovine-derived recombinant protein products are free of prions.

Another area of livestock research that will benefit from the technique is xenotransplantation. The demand for organs suitable for human transplantation far exceeds the current donor supply. Research in the area of xenotransplantation has focused on the development of transgenic pigs that express human genes13 or carry targeted mutations in detrimental genes14. The ability to create multiple genetic modifications could be used to disrupt active porcine endogenous retroviral sequences in the pig genome and eliminate cross-species retroviral infection (xenozoonosis). The potential to insert beneficial genes, mutate detrimental genes and eliminate the potential for xenozoonosis by sequential cloning are just a few of the potential applications of rapid, serial targeting.

Finally, sequential cloning in livestock may also benefit the development of large-animal models of human disease in areas where rodent models prove difficult to create or are ineffective at mimicking human disease phenotypes15.