cmv cre transgenic mice  (Jackson Laboratory)

Journal: Brain

Article Title: Imbalance of NRG1-ERBB2/3 signalling underlies altered myelination in Charcot–Marie–Tooth disease 4H

doi: 10.1093/brain/awac402

Figure Lengend Snippet: Generation and phenotypic characterization of a new mouse model of CMT4H. ( A – C ) Generation of a conditional knockout mouse model for CMT4H. ( A ) The floxed allele ( Fgd4 fl/+ ) was generated by flanking exon 4 with loxP sites (black arrowheads) in the Fgd4 gene leading to a frameshift by generation of a premature stop codon in exon 5. The conditional deletion of Fgd4 in SCs is induced by crossing Fgd4 fl/fl mice with mice expressing the cre recombinase under the Mpz/P0 promoter. For simplification the conditional knockout Fgd4 fl/fl ; P0cre mice are referred to as Fgd4 SC–/– . ( B ) Fgd4 transcript before ( top ) or after ( bottom ) excision of exon 4 following cre-recombinase expression. ( C ) Excision of the exon 4 from Fgd4 transcript was verified by RT-PCR using RNA extracted from the sciatic nerves of Fgd4 SC–/– and control mice. Due to exon 4 excision, the size of the amplified amplicon was 882 bp and 374 bp in WT and Fgd4 SC–/– sciatic nerves, respectively. ( D and E ) Loss of Fgd4/FRABIN alters myelination in vitro . ( D ) Number of myelin anomalies quantified in WT and Fgd4 SC–/– co-cultures. The presence of focal hypermyelination defects was evaluated in 150–160 myelinated segments per coverslip and three independent cultures. Myelinated segments were visualized by immunolabelling of MBP. Data are expressed as mean ± SEM. Statistical analysis: two-way ANOVA with Sidak post hoc test. ( E ) Illustration of myelinated segments in control and Fgd4 SC–/– co-cultures. Examples of myelin abnormalities that can be observed in the enlarged image and identified by asterisks. ( F ) Overexpression of Fgd4/FRABIN in Fgd4 SC–/– co-cultures reduces the proportion of abnormally myelinated fibres. Fgd4 SC–/– co-cultures were infected with Lv-EGFP or Lv-Fgd4, 1 day after seeding. Data are expressed as mean percentage ± SEM ( n = 4 independent cultures). Statistical analysis: unpaired Student’s t -test. ( G and H ) Loss of Fgd4/FRABIN alters PNS myelination in vivo . ( G ) Proportion of abnormal myelinated fibres in the distal part of the sciatic nerve of 3-, 6- and 18-month-old WT and Fgd4 SC–/– mice ( n = 3 animals per genotype). Data are expressed as mean percentage ± SEM. Statistical analysis: two-way repeated-measures ANOVA (group × time) with Sidak post hoc test. ( H ) Electron microscopy pictures illustrating myelination of sciatic nerves of WT ( left ) and Fgd4 SC–/– ( right ) mouse. Asterisks indicate outfoldings. Scale bar = 5 µm. ( I ) g-Ratio analysis revealed no statistical difference in myelin thickness in the sciatic nerve of at 3-, 6- and 18-month-old WT and Fgd4 SC–/– mice. A total of 500–1000 axons between 0.5 and 6 µm for animals ( n = 3 per genotype) were analysed. Data are presented as mean ± SEM ( n = 3 animals). ( J and K ) Maximum and mean forepaw intensities are significantly decreased in Fgd4 SC–/– mice compared to WT mice. Data are expressed as mean ± SEM ( n = 11 WT and n = 13 Fgd4 SC–/– mice). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For ubiquitous deletion of Fgd4 , Fgd4 fl/+ ; CMV-Cre , mice, so-called Fgd4 –/+ , were generated by crossing the floxed Fgd4 fl/+ line with transgenic mice expressing the Cre-recombinase under the CMV promoter [B6.C-Tg (CMV-cre)1Cgn/J], available at the Jackson Laboratories (#006054).

Techniques: Knock-Out, Generated, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, In Vitro, Over Expression, Infection, In Vivo, Electron Microscopy

Journal: Science Advances

Article Title: Neurexin-2: An inhibitory neurexin that restricts excitatory synapse formation in the hippocampus

doi: 10.1126/sciadv.add8856

Figure Lengend Snippet: ( A ) Nrxn2 cKO and constitutive KO strategy. Two selectable markers [puromycin (Puro) and neomycin (Neo)] were required to obtain embryonic stem cell clones with homologous recombination of the Nrxn2 gene. Exon 18, the first exon shared between Nrxn2 α and Nrxn2 β, was flanked by loxP sites, enabling Cre-mediated deletion of both Nrxn2 α and Nrxn2 β. ( B ) Nrxn2 cKO mice were bred with cytomegalovirus (CMV)–Cre mice to generate littermate wild-type (WT) and constitutive Nrxn2 KO mice. The constitutive Nrxn2 KO suppresses Nrxn2 mRNA levels but leaves Nrxn1 and Nrxn3 mRNA levels unchanged. The exon 18 Nrxn2 mRNA level measurements monitor the exon that is deleted, with the remaining 1% of mRNA detected likely because of background of quantitative reverse transcription polymerase chain reaction (RT-PCR) measurements. The decrease in the exon 23 mRNA levels is likely due to nonsense-mediated decay because the exon 18 deletion should not block Nrxn2 transcription, only the production of a functional protein. ( C to E ) Nrxn2 KO increases the AMPAR-mediated synaptic responses elicited by Schaffer collateral stimulation [(C) representative traces of AMPAR-EPSCs evoked by increasingly stronger stimuli and recorded at −70 mV in 50 μM picrotoxin and 50 μM D-D-AP-5 (2R)-amino-5-phosphonopentanoate) (2R)-amino-5-phosphonopentanoate); (D) input/output plot of the EPSC amplitude versus stimulus strength; (E) slope of the input/output relation]. ( F to H ) Nrxn2 KO increases NMDAR-mediated synaptic responses elicited by Schaffer collateral stimulation [same as (C) to (E) except that the responses were recorded at a holding potential of +40 mV in the presence of 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)]. ( I to K ) Nrxn2 KO has no effect on AMPAR-mediated synaptic responses elicited by stimulation of entorhinal cortex–derived axons [same as (C) to (E)]. Data are means ± SEM; the numbers of neurons per mice analyzed are listed in bar graphs. Statistical assessments were performed by the Mann-Whitney test comparing KO to control (B, E, H, and K) or by two-way analysis of variance (ANOVA) tests (D, G, and J), with * P < 0.05, ** P < 0.01, and *** P < 0.001. Ctrl., control.

Article Snippet: Cytomegalovirus (CMV)–Cre transgenic mice (Jackson Laboratory (JAX) stock number 006054) in which Cre is expressed throughout the whole organism ( ) were used to generate constitutive Nrxn2 KO mice by crossing the CMV-Cre driver mice with Nrxn2 f /f mice.

Techniques: Clone Assay, Homologous Recombination, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Functional Assay, Derivative Assay, MANN-WHITNEY, Control