Journal: Molecular and Cellular Biology

Article Title: Characterization of Mice with Targeted Deletion of Glycine Receptor Alpha 2

doi: 10.1128/mcb.00237-06

Figure Lengend Snippet: FIG. 1. Targeted mutation at the Glra2 locus. (A) Strategy for targeted deletion of Glra2 exons 6 and 7. Homologous recombination within a BAC containing exons 6 and 7 was used to replace exons 6 and 7 and the intervening intron with a PGK-neo cassette flanked by FRT sites. Shaded boxes labeled 1 represent probes used in Southern blot assays to screen for homologous recombination and deletion of exons 6 and 7. Wild-type (WT) genomic DNA digested with EcoRI and probed with probe 1 yields a fragment of 4.9 kb. In KO alleles, an endogenous EcoRI site present in the intron between exons 6 and 7 is deleted, yielding a 5.8-kb fragment when probed with probe 1. R, EcoRI. (B) Southern blot analysis of genomic DNA isolated from mouse tails. Southern blot assays of EcoRI-digested DNA probed with probe 1 presents a 4.9-kb band for the wild-type allele and a 3.5-kb band for the deletion-containing allele. (C) Diagram of GlyR2 pro- tein showing its four transmembrane domains (M1 to M4) with the approximate region of the protein encoded by exons 6 and 7 shaded in gray. Alternate splicing of exon 5 to exon 8 would result in a premature stop codon four amino acids into exon 8.

Article Snippet: Timed pregnant Glra2 KO mice and control C57/BL6 mice (Taconic, New York) at embryonic day 17 (E17) were used for all experiments, except where indicated otherwise.

Techniques: Mutagenesis, Homologous Recombination, Labeling, Southern Blot, Isolation

Journal: Molecular and Cellular Biology

Article Title: Characterization of Mice with Targeted Deletion of Glycine Receptor Alpha 2

doi: 10.1128/mcb.00237-06

Figure Lengend Snippet: FIG. 2. Glycine receptor expression in animals with Glra2 dis- rupted. (A) Reverse transcription-PCR analysis for Glra2 with RNA isolated from P0 mouse brain from wild-type (/), heterozygous (/), or homozygous Glra2 KO (/) littermates with primers spe- cific for Glra2 or ribosomal protein 17 (rps17). (B) Northern analyses were performed with total RNA (10 g) isolated from P0 mouse brain. The Glra2 probe was a 32P-labeled, full-length GlyR2 cDNA. A probe for -actin was used as a loading control. (C) In situ hybridiza- tion analysis of glycine receptor subunits in wild-type (WT) or Glra2 KO retinae at P0. The probes used recognized RNA species encoding glycine receptors alpha 1 (Glra1), alpha 2 (Glra2), alpha 3 (Glra3), alpha 4 (Glra4), and beta (GlrB).

Article Snippet: Timed pregnant Glra2 KO mice and control C57/BL6 mice (Taconic, New York) at embryonic day 17 (E17) were used for all experiments, except where indicated otherwise.

Techniques: Expressing, Reverse Transcription, Isolation, Northern Blot, Labeling, Control, In Situ

Journal: Molecular and Cellular Biology

Article Title: Characterization of Mice with Targeted Deletion of Glycine Receptor Alpha 2

doi: 10.1128/mcb.00237-06

Figure Lengend Snippet: FIG. 3. Analysis of P0 spinal cords from wild-type and Glra2 KO animals. (A) In situ hybridization for Glra2 RNA in cryosections of P0 spinal cord from wild-type (/) and Glra2 KO (/) animals. (B) Immunofluorescent staining for neurofilament 200 (red) and nu- clei counterstained with DAPI (blue) in P0 spinal cord sections.

Article Snippet: Timed pregnant Glra2 KO mice and control C57/BL6 mice (Taconic, New York) at embryonic day 17 (E17) were used for all experiments, except where indicated otherwise.

Techniques: In Situ Hybridization, Staining

Journal: Molecular and Cellular Biology

Article Title: Characterization of Mice with Targeted Deletion of Glycine Receptor Alpha 2

doi: 10.1128/mcb.00237-06

Figure Lengend Snippet: FIG. 4. Electrophysiological and calcium imaging responses to gly- cine (Gly), taurine (Tau), and GABA recorded in cortical pyramidal neurons. (A, B) Single-cell patch clamp recordings of E17 Glra2 KO mice. Current responses recorded in upper-layer pyramidal neurons (n 5/5) demonstrate that cortical neurons do not respond to 500 M glycine or 1 mM taurine in the presence (A) or absence (B) of 50 M bicuculline (Bic). However, these neurons exhibit normal GABA (30 M) responses (B). (C) Recordings of P7 Glra2 KO neurons (n 7/7) demonstrate normal responses to glycine and GABA. GABA re- sponses are abolished in the presence of 50 M bicuculline. (D, E) Calcium imaging of E17 coronal cortical slices from wild-type control (D) and Glra2 KO (E) animals. Calcium influx is induced in response to 500 M glycine (Gly) in wild-type (D) but not Glra2 KO (E) slices (n 2). Preincubation with strychnine (GlyS) inhibits glycine-in- duced calcium influx (D). Normal calcium influx in response to GABA is maintained in Glra2 KO slices. before, before stimulation.

Article Snippet: Timed pregnant Glra2 KO mice and control C57/BL6 mice (Taconic, New York) at embryonic day 17 (E17) were used for all experiments, except where indicated otherwise.

Techniques: Imaging, Patch Clamp, Control

Journal: Molecular and Cellular Biology

Article Title: Characterization of Mice with Targeted Deletion of Glycine Receptor Alpha 2

doi: 10.1128/mcb.00237-06

Figure Lengend Snippet: FIG. 5. Analysis of adult and neonatal cortexes from Glra2 KO animals. (A to D) Coronal sections of wild-type (WT) (A, C) and Glra2 mutant (B, D) P0 cortexes. Sections were stained with DAPI (A, B) or by in situ hybridization with a probe for GABA(A) receptor alpha 6 (C, D). (E to H) Hematoxylin-eosin staining of sections from adult (3-month-old) wild-type cortex (E) or cerebellum (G) or Glra2 mutant cortex (F) or cerebellum (H).

Article Snippet: Timed pregnant Glra2 KO mice and control C57/BL6 mice (Taconic, New York) at embryonic day 17 (E17) were used for all experiments, except where indicated otherwise.

Techniques: Mutagenesis, Staining, In Situ Hybridization

Journal: Molecular and Cellular Biology

Article Title: Characterization of Mice with Targeted Deletion of Glycine Receptor Alpha 2

doi: 10.1128/mcb.00237-06

Figure Lengend Snippet: FIG. 6. Expression of markers in wild-type and Glra2 mutant retinae at P0. In situ hybridization for TUG1 (A, B), Id2 (C, D), GABA(A) alpha 6 (E, F), Notch1 (G, H), Btg2 (I, J), and Nrl (K, L) in wild-type (A, C, E, G, I, K) or Glra2 KO (B, D, F, H, J, L) P0 retinae.

Article Snippet: Timed pregnant Glra2 KO mice and control C57/BL6 mice (Taconic, New York) at embryonic day 17 (E17) were used for all experiments, except where indicated otherwise.

Techniques: Expressing, Mutagenesis, In Situ Hybridization

Journal: Molecular and Cellular Biology

Article Title: Characterization of Mice with Targeted Deletion of Glycine Receptor Alpha 2

doi: 10.1128/mcb.00237-06

Figure Lengend Snippet: FIG. 7. Analysis of retinae of adult wild-type and Glra2 KO animals. Retinal sections of adult (3-month-old) wild-type (A, C) or Glra2 KO (B, D) animals are shown. (A, B) Retinal sections were immunostained for rhodopsin (RHO4D2, red), and nuclei were counterstained with DAPI (blue). (C, D) Hematoxylin-eosin stain- ing of adult retinal sections.

Article Snippet: Timed pregnant Glra2 KO mice and control C57/BL6 mice (Taconic, New York) at embryonic day 17 (E17) were used for all experiments, except where indicated otherwise.

Techniques: Staining