Generation of transgenic mouse fluorescent reporter lines for studying hematopoietic development

Abstract

During the development of the hematopoietic system, at least eight distinct lineages are generated in the mouse embryo. Transgenic mice expressing fluorescent proteins at various points in the hematopoietic hierarchy, from hematopoietic stem cell to multipotent progenitors to each of the final differentiated cell types, have provided valuable tools for tagging, tracking, and isolating these cells. In this chapter, we discuss general considerations in designing a transgene and survey available fluorescent probes and methods for confirming and analyzing transgene expression in the hematopoietic systems of the embryo, fetus, and postnatal/adult animal.

Figure 1. Basic design of a fluorescent transgenic construct

"R" marks the restriction enzyme sites used for removal of the bacterial backbone before transgene injection. s/pA represents the splicing and polyadenylation signals. A stop codon (denoted by "X") should also be included in the construct design. The enhancer may be positioned upstream or downstream from the promoter and sequences encoding the FP and regulatory signals. For additional details, see text.

Figure 2. Basic steps in the generation of a fluorescent transgenic reporter mouse line

Pronuclear injection of the transgene is shown in this cartoon but transgenic mouse lines can also be generated by blastocyst injection or embryo aggregation with genetically modified ES cells (see text).

Figure 3. Tagging of primitive and definitive erythroid lineages using GFP reporters

(A) GFP expression in the primitive erythroid cells of an embryonic stage (E)8.5 ε-globin-H2B-GFP embryo (24) (left panel, whole embryo, scale bar 200 μm; right panel, magnified view of yolk sac, scale bar, 50μm). Embryos were photographed on a Zeiss Lumar V12 stereomicroscope equipped with epifluorescence illumination and a NeoLumar S 1.5X FWD 30 mm objective. (B) Wet preparation of green fluorescent erythroid cells from the bone marrow (BM) and peripheral blood (PB) of an adult miR-144/451-GFP knock-in mouse (26). The cells were photographed on a Zeiss Axio Observer Z1 inverted microscope with epifluorescence illumination and a Plan-Apochromat 20X/0.8 objective. Scale bar, 20μm.