Journal: BMC Genomics

Article Title: A complete DNA sequence map of the ovine Major Histocompatibility Complex

doi: 10.1186/1471-2164-11-466

Figure Lengend Snippet: Assembly of 26 BAC-clone based DNA sequences covering entire Ovine MHC region

Article Snippet: Shotgun sequencing libraries were constructed individually for each of the 26 BAC clones following the modified protocols described by Celera Genomics Group [ ].

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Rosiglitazone-Induced Mitochondrial Biogenesis in White Adipose Tissue Is Independent of Peroxisome Proliferator-Activated Receptor γ Coactivator-1α

doi: 10.1371/journal.pone.0026989

Figure Lengend Snippet: (A) A targeting vector, containing a PGK-NEO selection cassette flanked by FRT sites in intron 5 and having exons 4 and 5 of Ppargc1 a gene flanked by loxP sites, was used to generate mice with floxed Ppargc1a alleles. PGC-1α-FAT-KO mice were generated by crossing mice with floxed Ppargc1a alleles to aP2-Cre mice that overexpress Cre recombinase in adipose tissues under the control of the adipocyte-specific aP2 promoter. (B) PCR of tail genomic DNA was used to detect loxP flanked Ppargc1a alleles and the presence of aP2-Cre transgene. Mice homozygous for the loxP-flanked allele and positive for the aP2-Cre transgene are referred to as fat-specific PGC-1α knockout mice (PGC-1α-FAT-KO). (C) Expression of PGC-1α mRNA was analyzed by real-time quantitative PCR in several tissues of Wt (white bars) or PGC-1α-FAT-KO (black bars) mice. Values are mean ± SEM. ** P <0.01, n = 4–5 animals/group.

Article Snippet: To generate mice with floxed Ppargc1a alleles, a targeting vector was constructed by subcloning a Bste II - SnaB I (7,491 bp, containing exons 3, 4 and 5) and a SnaB I – Xho I (3,709 bp of intron 5) DNA fragment of a BAC genomic DNA clone encoding the murine Ppargc1a gene locus (Incyte Genomics, Palo Alto, CA) upstream and downstream, respectively, of a PGK-neomycin cassette flanked by two FRT sites and one LoxP site (gift of U. Mueller, TSRI, La Jolla, CA).

Techniques: Plasmid Preparation, Selection, Generated, Control, Knock-Out, Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of General Physiology

Article Title: The Light Peak of the Electroretinogram Is Dependent on Voltage-gated Calcium Channels and Antagonized by Bestrophin (Best-1)

doi: 10.1085/jgp.200509473

Figure Lengend Snippet: Targeted disruption of the Vmd2 gene. A schematic diagram of the wild-type (wt) locus, targeting vector, and mutant (mt) locus is presented in A. Thick lines represent fragments used for constructing the targeting vector 5′ and 3′ arms. Numbered solid boxes depict Vmd2 exons. The neo r and DTA gene expression cassettes are indicated by the labeled gray boxes. The drawing line represents plasmid vector sequence. The external 3′ probe used in B is indicated beneath the mt locus. Restriction enzyme sites: Bg, BglI; E, EcoRI; Hc, HincII; A, AvrII; X, XbaI; B, BamHI. Southern blot analysis of wt ( Vmd2 + / + ), heterozygous ( Vmd2 + / − ), and homozygous (V md2 − / − ) mouse tail genomic DNA digested with HincII (B and C). The wt fragment is 12.7 kb and the mt fragment is 10.8 kb. RT-PCR analysis of total RNA was used to confirm a null phenotype (D). The 315-bp Vmd2 -derived PCR product was present in Vmd2 + / + and Vmd2 + / − mice but absent in the −/− lane. In the positive control, a 428-bp product was obtained from all mice using primers for fibulin-3. Best-1 was detected by immunohistochemistry in the RPE of Vmd2 + / + but not Vmd2 − / − mice (E). Arrows in E indicate the RPE.

Article Snippet: A BAC clone containing the entire mouse genomic DNA for Vmd2 was isolated by screening a genomic 129/SvJ mouse library (Incyte Genomics).

Techniques: Disruption, Plasmid Preparation, Mutagenesis, Gene Expression, Labeling, Sequencing, Southern Blot, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Positive Control, Immunohistochemistry

Journal: The Journal of General Physiology

Article Title: The Light Peak of the Electroretinogram Is Dependent on Voltage-gated Calcium Channels and Antagonized by Bestrophin (Best-1)

doi: 10.1085/jgp.200509473

Figure Lengend Snippet: Effect of Vmd2 disruption on the flash ERG. Amplitude (A) and implicit time (B) of the a-wave and b-wave components of the dark-adapted strobe flash ERG plotted as a function of stimulus intensity. Symbols indicate the mean ± SEM result of six mice.

Article Snippet: A BAC clone containing the entire mouse genomic DNA for Vmd2 was isolated by screening a genomic 129/SvJ mouse library (Incyte Genomics).

Techniques: Disruption

Journal: The Journal of General Physiology

Article Title: The Light Peak of the Electroretinogram Is Dependent on Voltage-gated Calcium Channels and Antagonized by Bestrophin (Best-1)

doi: 10.1085/jgp.200509473

Figure Lengend Snippet: Effect of Vmd2 disruption on RPE-generated ERG components. ERGs were recorded to 7-min duration stimulus flashes from Vmd2 +/+ (blue) and Vmd2 −/− (red) mice. Grand average of responses obtained from ≥15 individual mice are shown in A for each stimulus intensity. Stimulus presentation is represented by the lower trace (black). Intensity–response functions (B) for the amplitude of the major ERG components generated by the RPE in response to light. Data points indicate the mean ± SEM of >15 responses obtained from different mice.

Article Snippet: A BAC clone containing the entire mouse genomic DNA for Vmd2 was isolated by screening a genomic 129/SvJ mouse library (Incyte Genomics).

Techniques: Disruption, Generated

Journal: The Journal of General Physiology

Article Title: The Light Peak of the Electroretinogram Is Dependent on Voltage-gated Calcium Channels and Antagonized by Bestrophin (Best-1)

doi: 10.1085/jgp.200509473

Figure Lengend Snippet: Cl − conductances in RPE cells isolated from Vmd2 +/+ and Vmd2 −/− mice. RPE cells were isolated from mice as described in the Materials and Methods. As reported by , apical microvilli could be distinguished from the basal surface of the cells as shown in A. Cl − currents in response to a series of step potentials (B) were analyzed by whole cell patch-clamp in low (10 nM) or high (400 nM) Ca 2+ . Examples of recordings obtained from individual cells in high Ca 2+ are shown in C for both Vmd2 +/+ and Vmd2 −/− mice. I/V plots for low and high Ca 2+ conditions are shown in D for both Vmd2 +/+ and Vmd2 −/− mice (mean ± SD, 6 ≤ n ≤ 22 cells). Note that larger currents were obtained in high Ca 2+ conditions. A comparison of currents obtained in high Ca 2+ conditions (E) indicates no differences between Vmd2 +/+ and Vmd2 −/− mice.

Article Snippet: A BAC clone containing the entire mouse genomic DNA for Vmd2 was isolated by screening a genomic 129/SvJ mouse library (Incyte Genomics).

Techniques: Isolation, Patch Clamp, Comparison

Journal: The Journal of General Physiology

Article Title: The Light Peak of the Electroretinogram Is Dependent on Voltage-gated Calcium Channels and Antagonized by Bestrophin (Best-1)

doi: 10.1085/jgp.200509473

Figure Lengend Snippet: Effect of Ca 2+ channel blocker nimodipine on RPE-generated ERG components. The effect of nimodipine on the DC-ERG of Vmd2 +/+ mice was determined 30 min after intraperitoneal injection of a 1 mg/kg dose of nimodipine using a 2 log cd/m 2 stimulus. Representative ERGs (A) obtained after injection of vehicle alone (control) or 1 mg/kg nimodipine. Intensity–response functions for major ERG components are shown in B. Each data point indicates the average (±SD) for three mice. The amplitude of the a-wave was measured 8 ms after stimulus presentation. The amplitude of the b-wave was measured to the peak of the b-wave from the a-wave trough or, if no a-wave was present, from the baseline. C shows grand average dc-ERG waveforms generated from animals receiving nimodipine or vehicle alone ( n = 8 both groups). Note the obvious reduction in amplitude of the LP in nimodipine-treated mice. C-wave, FO, LP, and off-response amplitudes are shown in D. Bars represent mean ± SEM.

Article Snippet: A BAC clone containing the entire mouse genomic DNA for Vmd2 was isolated by screening a genomic 129/SvJ mouse library (Incyte Genomics).

Techniques: Generated, Injection, Control

Journal: The Journal of General Physiology

Article Title: The Light Peak of the Electroretinogram Is Dependent on Voltage-gated Calcium Channels and Antagonized by Bestrophin (Best-1)

doi: 10.1085/jgp.200509473

Figure Lengend Snippet: Effect of nimodipine on RPE-generated ERG components of Vmd2 −/− mice. The effect of nimodipine on the DC-ERG of Vmd2 −/− mice was determined as indicated for Vmd2 +/+ mice in using stimuli of 2 log cd/m 2 (A and B, n = 8) or −2 log cd/m 2 (C and D, n = 5). Examination of grand average waveforms (A and C) indicates that the most obvious effects are on the amplitude of the LP. Examination of maximum response amplitudes (B and D) confirms that there is minimum effect of nimodipine on the c-wave, FO, or off-response. However, the LP was significantly (*, P < 0.05) diminished at 2 log cd/m 2 (B). Although the LP was reduced at −2 log cd/m 2 (D), the effect was of borderline significance (P = 0.09). Data in C and D are mean ± SEM.

Article Snippet: A BAC clone containing the entire mouse genomic DNA for Vmd2 was isolated by screening a genomic 129/SvJ mouse library (Incyte Genomics).

Techniques: Generated

Journal: The Journal of General Physiology

Article Title: The Light Peak of the Electroretinogram Is Dependent on Voltage-gated Calcium Channels and Antagonized by Bestrophin (Best-1)

doi: 10.1085/jgp.200509473

Figure Lengend Snippet: Heart Rates (Beats per Minute) Determined before and after dc-ERG Recordings

Article Snippet: A BAC clone containing the entire mouse genomic DNA for Vmd2 was isolated by screening a genomic 129/SvJ mouse library (Incyte Genomics).

Techniques: Control

Journal: The Journal of General Physiology

Article Title: The Light Peak of the Electroretinogram Is Dependent on Voltage-gated Calcium Channels and Antagonized by Bestrophin (Best-1)

doi: 10.1085/jgp.200509473

Figure Lengend Snippet: Effect of best-1 on changes in [Ca 2+ ] I in response to ATP stimulation. RPE sheets were isolated from the eyes of Vmd2 +/+ or Vmd2 −/− mice (A) and loaded with fura-2. Changes in [Ca 2+ ] I were followed in response to stimulation with 100 μM ATP. Examples of data from individual cells are shown in B, where the red tracing is from cells isolated from Vmd2 −/− mice and the blue tracing is from cells isolated from Vmd2 +/+ littermates. C indicates the maximum average percent change in [Ca 2+ ] I (average ± SD, n = 13 for Vmd2 +/+ , n = 7 for Vmd2 −/− ). **, P < 0.05.

Article Snippet: A BAC clone containing the entire mouse genomic DNA for Vmd2 was isolated by screening a genomic 129/SvJ mouse library (Incyte Genomics).

Techniques: Isolation