Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells

Abstract

Stem cells represent a unique population of cells with self-renewal capacity. Although they are important therapeutic targets, the genetic manipulation of tissue-specific stem cells has been limited, which complicates the study and practical application of these cells. Here, we demonstrate successful gene trapping and homologous recombination in spermatogonial stem cells. Cultured spermatogonial stem cells were transfected with gene trap or gene targeting vectors. Mutagenized stem cells were expanded clonally by drug selection. These cells underwent spermatogenesis and produced heterozygous offspring after transplantation into the seminiferous tubules of infertile mouse testes. Heterozygous mutant mice were intercrossed to produce homozygous gene knockouts. Using this strategy, the efficiency of homologous recombination for the occludin gene locus was 1.7% using a nonisogenic DNA construct. These results demonstrate the feasibility of altering genes in tissue-specific stem cells in a manner similar to embryonic stem cells and have important implications for gene therapy and animal transgenesis.spermatogenesis ͉ germ cell ͉ testis ͉ transplantation S tem cells represent a unique cell population with self-renewal potential (1). Although stem cells are low in number, these cells proliferate extensively to sustain the various self-renewing tissues, such as bone marrow and intestine. Although these tissue-specific stem cells normally divide very slowly, stem cells are the last cell type to be destroyed after cytotoxic damage, and they regenerate the entire tissue in a relatively short time. In addition, stem cells often have migratory activities, and they can be transplanted between animals; transplanted stem cells migrate to a specific niche and regenerate the self-renewing tissue. Because of their unique properties, stem cells have become the attractive target of cell and gene therapies.Among the many types of tissue-specific stem cells, spermatogonial stem cells are unique in that they have germ-line potential (2, 3). Genetic modification of spermatogonial stem cells creates permanent changes in the germ line, which are transmitted to the offspring by means of fertilization. In contrast to female germ-line cells, which cease to divide after birth, male germ-line cells proliferate continuously and produce sperm throughout the life of the animal. If these stem cells could be cultured and manipulated in a manner similar to embryonic stem (ES) cells (4, 5), they could be used to create knockout animals. As a first step toward this goal, a germ cell transplantation technique was developed in 1994 (6, 7), in which dissociated donor testis cells colonized the seminiferous tubules of infertile recipient testis and produced donor-derived spermatogenesis and offspring. Although this technique was an opportunity to produce offspring from manipulated spermatogonial stem cells, it has been difficult to produce transgenic animals using spermatogonial stem cells, because their number is very low in the testis, and the lack of me...

Behavior of mouse spermatogonial stem-like cells on an electrospun nanofibrillar matrix

“…The numbers of spermatogonial stem-like cells that migrated to the seminiferous basal membrane and donor-derived colonies of spermatogenesis in each recipient testis were counted using a fluorescent microscope (30 seminiferous tubules were randomly selected). Because each colony was derived from a single SSC [19,24], we counted the donor-derived colonies of spermatogenesis from the 30 randomly selected seminiferous tubules and compared them in two different treatments. This provided a relative quantification of spermatogonial stem-like cell number in an injected cell suspension.…”

In vitro production of fertile sperm from murine spermatogonial stem cell lines

“…The cultured SSCs, termed germline stem (GS) cells or SSC lines, proliferated in a self-renewing manner for up to or beyond 2 years, while remaining stable genetically, epigenetically and karyotypically 10 . It has been reported that genetic modifications were feasible in GS cells 11,12 . Nonetheless, it has not yet been possible to induce spermatogenesis from these cells in vitro.…”

Brief History, Pitfalls, and Prospects of Mammalian Spermatogonial Stem Cell Research

“…The level of efficiency is five to ten times higher than that achieved by conventional methods using eggs or oocytes (Nagano et al 2001b). Moreover, the frequency of homologous recombination is comparable to that achieved in ES cells (Kanatsu-Shinohara et al 2006c). Thus, SSCs may be used as a vehicle for gene targeting.…”

“…This is in contrast to ES cells, which often lose their germ cell potential due to trisomy (Liu et al 1997;Longo et al 1997). Similar to ES cells, GS cells can be used to produce transgenic and knockout animals via genetic transduction and drug selection (Kanatsu- Shinohara et al 2005aShinohara et al , 2006c. However, the efficiency of transgenesis is about 50%, which reflects the fact that the transgene is transmitted to half of the haploid cells.…”