A Noninvasive Genetic/Pharmacologic Strategy for Visualizing Cell Morphology and Clonal Relationships in the Mouse

Abstract

Analysis of cellular morphology is the most general approach to neuronal classification. With the increased use of genetically engineered mice, there is a growing need for methods that can selectively visualize the morphologies of specified subsets of neurons. This capability is needed both to define cell morphologic phenotypes and to mark cells in a noninvasive manner for lineage studies. To this end, we describe a bipartite genetic system based on a Cre-estrogen receptor (ER) fusion protein that irreversibly activates a plasma membrane-bound alkaline phosphatase reporter gene by site-specific recombination. Because the efficiency and timing of gene rearrangement is controlled pharmacologically, a sparse subset of labeled cells can be generated from the set of CreER-expressing cells at any time during development. Histochemical visualization of alkaline phosphatase activity reveals neuronal morphology with strong and uniform labeling of all processes.

Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication

“…MEFs were prepared as described previously (31). Embryo genotypes were Rnaseh1 fl/fl Gt(ROSA)26Sor tm1(Cre/Esr1)Nat homozygous (32). Conditional gene knockout in murine cells was via the action of cre-recombinase on LoxP sites flanking exons V-VII of the Rnaseh1 gene and confirmed using an in-gel activity assay for RNase H (33).…”

Axonal Growth and Guidance Defects inFrizzled3Knock-Out Mice: A Comparison of Diffusion Tensor Magnetic Resonance Imaging, Neurofilament Staining, and Genetically Directed Cell Labeling

“…Pregnant females were given a single intraperitoneal injection of 200 g of 4-hydroxytamoxafen (4HT) in sunflower seed oil between 10 and 13 d of gestation. Tissue processing and AP histochemistry were performed essentially as described by Badea et al (2003) using 300 m vibratome sections of immersion-fixed brains. A total of eight pairs of Fz3 Ϫ/Ϫ and wild-type (WT) littermates were analyzed by AP staining.…”

“…The second genetic marking method used here combines a ubiquitously expressed CreER recombinase, a Cre-activated AP reporter, and low-dose 4HT injection to generate sparse labeling of neurons (Badea et al, 2003). By preparing coronal sections of Ϫ/Ϫ .…”

“…When 4HT exposure occurs during the period of active neuronal proliferation (before approximately E15 for cerebral cortical neurons), AP activity is seen in a small clusters of cells, with each cluster representing the clonal progeny of individual precursors in which a recombination event occurred. Because the number of initiating events is determined by 4HT dose and the size of the resulting clones is determined by the timing of 4HT exposure (with earlier exposure leading to larger clones) (Badea et al, 2003), one can readily produce a brain in which many small clusters of labeled neurons efficiently sample the major structures.…”

“…One method uses a yellow fluorescent protein (YFP) transgene driven by the Thy1 promotor (Feng et al, 2000). The second method uses pharmacologic activation of site-specific DNA recombination in transgenic mice to produce a sparse population of cells expressing human placental alkaline phosphatase (AP) (the CreER;ZAP system) (Badea et al, 2003).…”