Genome engineering via homologous recombination in mouse embryonic stem (ES) cells: an amazingly versatile tool for the study of mammalian biology

The ability to introduce genetic modifications in the germ line of complex organisms has been a long-standing goal of those who study developmental biology. In this regard, the mouse, a favorite model for the study of the mammals, is unique: indeed not only is it possible since the late seventies, to add genes to the mouse genome like in several other complex organisms but also to perform gene replacement and modification. This has been made possible via two technological breakthroughs: 1) the isolation and culture of embryonic stem cells (ES), which have the unique ability to colonize all the tissues of an host embryo including its germ line; 2) the development of methods allowing homologous recombination between an incoming DNA and its cognate chromosomal sequence (gene ''targeting''). As a result, it has become possible to create mice bearing null mutations in any cloned gene (knock-out mice). Such a possibility has revolutionized the genetic approach of almost all aspects of the biology of the mouse. In recent years, the scope of gene targeting has been widened even more, due to the refinement of the knock-out technology: other types of genetic modifications may now be created, including subtle mutations (point mutations, micro deletions or insertions, etc.) and chromosomal rearrangements such as large deletions, duplications and translocations. Finally, methods have been devised which permit the creation of conditional mutations, allowing the study of gene function throughout the life of an animal, when gene inactivation entails embryonic lethality. In this paper, we present an overview of the methods and scenarios used for the programmed modification of mouse genome, and we underline their enormous interest for the study of mammalian biology

Effect of the absence of heat shock protein 70.1 (hsp70.1) on retinal photoreceptors in normal and rd mice

The purposes of this study are to elucidate the retinal changes of heat shock protein 70.1 (hsp70.1) knockout mice and to compare them between in normal and in retinal degeneration (rd) mice. Eyes of hsp70.1 wild type (+/+) and knockout (-/-) mice in the C57BL/6 or FVB genetic backgrounds respectively, which were reared in the normal environment, were examined by fundus photography, electroretinography, light microscopy, terminal dUTP nick-end labeling (TUNEL) stain, and immunohistochemistry. In C57BL/6 mice, fundus photography showed no changes between hsp70.1+/+ and -/- mice at 1 and 6 months of age. Electroretinographic examination showed a tendency of decreased amplitude of a- and b-wave with aging in both genotype, but there were not different statistically. The ratios of the thickness of inner nuclear and outer nuclear layer to the retinal thickness were respectively decreased with aging in both genotype, but there were not different statstically. TUNEL assay showed a few positively labeled cells in the ganglion cell, inner nuclear and outer nuclear layers and the immunohistochemistry showed no immunopositivity of hsp70 in the inner segments of photoreceptor cell layer in both genotype. In rd mice, fundus photography showed a narrowing of the retinal vessels at the age of 4 weeks, however, there were no differences of retinal changes including pigment epithelial layer in both genotype. Electroretinographic examination at the postnatal 2, 3 and 4 weeks showed no differences between them. Loss of photoreceptor cell and outer nuclear layers showed no differences in both genotype. In conclusion, there were no differences of the retinal changes at least under the normal environmental condition in hsp70.1+/+ and -/- mice. These results show that hsp70.1-/- mice can be used to study the role of hsp70.1 to the external stress to the retina.