Subregion- and Cell Type–Restricted Gene Knockout in Mouse Brain

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Subregion- and Cell Type–Restricted Gene Knockout in Mouse Brain Joe Z Tsien, Dong Feng Chen, David Gerber, Cindy Tom, Eric H Mercer, David J Anderson, Mark Mayford, Eric R Kandel, Susumu Tonegawa Cell Volume 87, Issue 7, Pages 1317-1326 (December 1996) DOI: 10.1016/S0092-8674(00)81826-7 Copyright © 1996 Cell Press Terms and Conditions

Figure 1 The Cre/loxP System for Gene Knockout (A) Strategy for cell type–restricted gene knockout. (B) In situ detection of the Cre recombination by lacZ expression. Cell 1996 87, 1317-1326DOI: (10.1016/S0092-8674(00)81826-7) Copyright © 1996 Cell Press Terms and Conditions

Figure 2 Plasmid Constructs (A) Schematic representation of a construct pJT-Cre for production of Cre transgenic mouse. (B) Schematic representation of a construct pcAct-XstopXlacZ for production of reporter mouse. Cell 1996 87, 1317-1326DOI: (10.1016/S0092-8674(00)81826-7) Copyright © 1996 Cell Press Terms and Conditions

Figure 3 Northern Blot Analysis of Cre mRNA Expression in the Adult Brain from Transgenic Lines (A) Cre mRNA expression in adult forebrain (FB) regions (cortex and hippocampus) and hindbrain (HB) regions (cerebellum and brain stem) from mouse lines T17, T29, T35, T40, T44, and T58. Twenty micrograms of total RNA per lane was loaded and blotted. The size of Cre mRNA is indicated at right. (B) Cre mRNA expression in adult cortex (CX), hippocampus (HP), and cerebellum (CB) from brains of mouse lines T17, T29–1, and T29–2. The expected 2.6 kb Cre mRNA is indicated. (C) Expression of Cre mRNA in the developing hippocampus of T29–1 at P16, P19, P23, and adult (Ad). In (B) and (C) 10 μg/lane total RNA was loaded and blotted as described in Experimental Procedures. The same blot was reprobed with G3PDH probe as a control for the amount of RNA loaded. G3PDH, glycerol-3-phosphate dehydrogenase. Cell 1996 87, 1317-1326DOI: (10.1016/S0092-8674(00)81826-7) Copyright © 1996 Cell Press Terms and Conditions

Figure 4 Cre/loxP Recombination Patterns in the Brains from Different Cre/lacZ Transgenic Lines Determined by X-Gal Blue Staining Sagittal brain sections of the lacZ reporter (R), T17/lacZ, T29–1/lacZ, and T29–2/lacZ mice were stained with X-Gal and then counterstained with cresyl violet (A, C, E, G) or with eosin (B, D, F, H). In cresyl violet–stained sections, cerebellum and hippocamal neurons are heavily stained, partially obscuring the X-Gal staining, which is restricted to different regions of the forebrain in all lines illustrated (see eosin-stained sections). Scale bar, 1 mm. Cell 1996 87, 1317-1326DOI: (10.1016/S0092-8674(00)81826-7) Copyright © 1996 Cell Press Terms and Conditions

Figure 5 Distribution and Efficiency of Cre/loxP Recombination in Various Transgenic Mice Examined at Higher Magnification (A, C, E, and G) The distribution of Cre/loxP recombination in the hippocampus of the reporter T17/lacZ, T29–1/lacZ, and T29–2/lacZ mice, respectively, as visualized by X-Gal staining and cresyl violet counterstaining. Scale bar, 500 μm. (B, D, F, and H) The CA1 region of the hipppocampus from the same set of mice. Scale bar, 100 μm. (I) The cortex and striatum of T29–1/lacZ. Scale bar, 500 μm. (J) The cortex of T29–2/lacZ. Scale bar, 200 μm. Cell 1996 87, 1317-1326DOI: (10.1016/S0092-8674(00)81826-7) Copyright © 1996 Cell Press Terms and Conditions

Figure 6 Immunohistochemistry of Cre Protein in the Brains from wild-type, T29–1, and T29–2 Mice (A) Hippocampus and cortex (CX) of the wild-type mice, stained with the Cre monoclonal antibody. (B) Hippocampus and cortex of T29–1. DG, Dentate gyrus. (C) Striatum (STR) region of T29–2. (D) Hippocampus and cortex of T29–2. (E) Cerebellar (CB) region of T29–2. Cell 1996 87, 1317-1326DOI: (10.1016/S0092-8674(00)81826-7) Copyright © 1996 Cell Press Terms and Conditions

Figure 7 Developmental Time Course of Cre/loxP Recombination in Three Cre Transgenic Mice Individually Crossed to the lacZ reporter Mice The brain sections were stained with X-Gal and counterstained with eosin. The types and postnatal dates of the mice are indicated. (A–H and J) The hippocampus. (I and K) The cortex and striatum. DG, dentate gyrus; CX, cortex; Str, striatum. Scale bar, 500 μm. Cell 1996 87, 1317-1326DOI: (10.1016/S0092-8674(00)81826-7) Copyright © 1996 Cell Press Terms and Conditions