Gene-targeted Cells Research Articles

5 The Use of Embryonic Stem Cells for the Genetic Manipulation of the Mouse

Publisher Summary This chapter describes methods for the design and isolation of gene-targeted and gene-trapped embryonic stem (ES) cell lines. In addition, morula aggregation, a procedure alternative to the classic microinjection of ES cells into the blastocysts, is described, and its performance is discussed in comparison with microinjection methods. Adult mice develop normally from these embryos and ES cells contribute to all tissues, including the germline. The unique characteristics allow the introduction of foreign DNA, the screening for rare integration events in ES cells, and the generation of ES cell-derived mouse strains. This strategy has been successfully used for gene targeting by homologous recombination, which has become a powerful tool to study gene function. By this procedure, designed mutations are introduced into particular genes, and mouse lines carrying the mutated allele are generated. In addition, ES cells have been used for the random mutagenesis of the mouse genome by the insertion of gene and enhancer trap vectors.

Unimpaired macrophage differentiation and activation in mice lacking the zinc finger transplantation factor NGFI-A (EGR1).

The zinc finger protein NGFI-A (also called EGR1, Krox24, or zif268) is a candidate regulator of myeloid cell differentiation. Evidence supporting this hypothesis is twofold. First, NGFI-A antisense oligonucleotides prevent macrophage differentiation in HL-60 and U937 myeloid leukemia cell lines and in normal bone marrow cells. Second, enforced expression of NGFI-A blocks granulocytic differentiation and promotes macrophage differentiation in HL-60 cells and in the hematopoietic progenitor cell line 32D. We sought to determine the effect of NGFI-A deficiency on macrophage differentiation and function in vivo by examining native bone marrow cells from mice homozygous for a disrupted allele of NGFI-A derived from gene-targeted ES cells. Macrophages were observed in peripheral blood and several tissues, indicating that NGFI-A was not required for the formation of a variety of macrophage compartments. No differences in myeloid cell differentiation were observed between wild-type and NGFI-A-/- bone marrow cells cultured in the presence of macrophage, granulocyte-macrophage, or granulocyte colony-stimulating factor (M-CSF, GM-CSF, or G-CSF). Activation of NGFI-A-/- macrophages was comparable to that of wild-type macrophages as determined by nitric oxide production and increased cell surface expression of class II major histocompatibility complex molecules. Moreover, NGFI-A-/- mice showed no increased mortality or bacteria] burden when challenged with Listeria monocytogenes. Together, these results indicate that NGFI-A is not required for macrophage differentiation or activation.