Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells.

A novel glass needle-mediated microinjection method for delivery of macromolecules, including proteins and larger transgene DNAs, into the nuclei of blood stem/progenitor cells was developed. Temporary immobilization of cells to extracellular matrix-coated dishes has enabled rapid and consistent injection of macromolecules into nuclei of CD34(+), CD34(+)/CD38(-), and CD34(+)/CD38(-)/Thy-1(lo) human cord blood cells. Immobilization and detachment protocols were identified, which had no adverse effect on cell survival, progenitor cell function (colony forming ability), or stem cell function (NOD/SCID reconstituting ability).

Repetitive endocytosis and recycling of the beta 2-adrenergic receptor during agonist-induced steady state redistribution.

The human beta 2-adrenergic receptor (beta 2AR) rapidly internalizes after binding agonist, resulting in a dramatic redistribution of receptors from the plasma membrane and into endocytic vesicles. We sought to determine whether intracellular receptors constitute a static pool or represent a fraction of dynamically internalizing and recycling receptors. Using cells expressing a beta 2AR with an epitope tag at its amino-terminal ectodomain, changes in surface receptor levels were measured by flow cytometry and radioligand binding assays.

Ligand-stimulated beta 2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes.

The small GTPase rab5 appears to be rate-limiting for the constitutive internalization of transferrin receptor and for fluid-phase endocytosis. However, it is unknown whether rab5 regulates receptors whose internalization is stimulated by the binding of ligand, and whether such receptors change the underlying rate of the endocytic pathways they utilize. As a model for ligand-stimulated endocytosis, we used transfected HEK293 cells expressing high levels of an epitope-tagged human beta 2-adrenergic receptor.