Cattle call for gene targeting

Abstract

The discovery that a differentiated somatic cell can give rise to a new organism through nuclear transfer cloning touched off a revolution in genetics. A new study outlining how to target genes serially in cows ushers in a new era in large-animal genetics. N E W S A N D V I E W SNATURE GENETICS VOLUME 36 | NUMBER 7 | JULY 2004 671Somatic 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 cell 1 . Cloning using somatic cells cultured long-term 2 laid the foundation for single-gene targeting in species other than mouse 3 . 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 colleagues 4 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). Rejuvenation by serial cloningGenerating 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 (F 1 ) male and female heterozygous calves, which would be crossbred 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