1. Volume 19, Issue 6, Pages 1285-1296 (December 1997)
Knockout of the Vesicular Monoamine Transporter 2 Gene Results in Neonatal Death and Supersensitivity to Cocaine and Amphetamine 
Yan-Min Wang, Raul R Gainetdinov, Fabio Fumagalli, Fei Xu, Sara R Jones, Cheryl B Bock, Gary W Miller, R.Mark Wightman, Marc G Caron 
Neuron 
Volume 19, Issue 6, Pages (December 1997)
DOI: /S (00)

2. Figure 1 Maps of the Targeting Vector and the Mock Construct
The mouse genomic fragment clone 11 was isolated from a Stratagene 129 SvJ library by standard colony hybridization using a PCR probe from the 5′ end of rat cDNA. The restriction site abbreviations are: H, HindIII; N, NotI; Sc, SacI; Sn, SnaI; X, XbaI; and Xh, XhoI. The region between HindIII and SnaI on clone 11 containing the coding sequence for transmembrane domains 3 and 4 of VMAT2 was deleted and replaced with PGK-neo. The 3′ XbaI fragment of clone 11 was reserved as an external probe for Southern analysis. To facilitate PCR screening of embryonic stem cell clones, a mock construct containing the SnaI/XbaI fragment and part of the neomycin cassette was made as a positive control. pPNT and pGEM4Z are the two vectors used to construct the knockout vector and mock vector, respectively. Abbreviations: WT, wild-type littermate; KO, knockout.
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3. Figure 2 Confirmation of VMAT2 Gene Deletion
(A) Southern blot analysis of mouse genomic DNA. The Southern blot was prepared with 15 μg of genomic DNA per lane and probed with a 1.4 kb 3′ external genomic fragment, as indicated in Figure 1. Abbreviations: +/+, wild-type littermate; +/−, heterozygote; −/−, homozygote.
(B) RT–PCR analysis of mouse brain poly (A+) RNA. Poly (A+) RNA (0.5 μg) was used for each reverse transcription assay. Equal volume of cDNA templates was used for each PCR assay. The PCR primers used flank the neomycin cassette (see Figure 1), for the purpose of detecting potential readthrough from the neomycin cDNA. The heterozygote (+/−) has a reduced amount of transcripts as compared to the wild-type littermate (+/+), while the homozygote (−/−) is devoid of transcription of VMAT2 (top). G3PDH (983 bp) was used as internal control (bottom).
(C) Western blot analysis of whole brain synaptic vesicles. Samples (25 μg) of vesicles were solubilized and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 14%), transferred to nitrocellulose, subjected to Western blot analysis with anti-VMAT2-Ct (top) or anti–α-tubulin (bottom) antibodies, and developed with chemiluminescence. Molecular mass markers (kDa) are shown on the left. To confirm equal loading and transfer of proteins, the blots were stripped and reprobed with an antibody to α-tubulin. No difference in the level of α-tubulin immunoreactivity was observed between wild-type littermate and heterozygous VMAT2 knockout as determined by densitometric analysis using internal dilutional standards from wild-type littermate brain.
(D) Tetrabenazine (TBZ) binding to brain vesicle preparations. Newborn whole brains were used to obtain vesicle preparations. Increasing concentrations of TBZ were used to inhibit binding of the tritiated analog 3H-dihydrotetrabenazine (1 nM). Each point represents the mean of three binding experiments performed in triplicate with separate brain vesicle preparations. The homozygote binding parallels that of nonspecific binding. KD = 0.1 nM and Bmax = 12 fmol/mg tissue.
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4. Figure 2 Confirmation of VMAT2 Gene Deletion
(A) Southern blot analysis of mouse genomic DNA. The Southern blot was prepared with 15 μg of genomic DNA per lane and probed with a 1.4 kb 3′ external genomic fragment, as indicated in Figure 1. Abbreviations: +/+, wild-type littermate; +/−, heterozygote; −/−, homozygote.
(B) RT–PCR analysis of mouse brain poly (A+) RNA. Poly (A+) RNA (0.5 μg) was used for each reverse transcription assay. Equal volume of cDNA templates was used for each PCR assay. The PCR primers used flank the neomycin cassette (see Figure 1), for the purpose of detecting potential readthrough from the neomycin cDNA. The heterozygote (+/−) has a reduced amount of transcripts as compared to the wild-type littermate (+/+), while the homozygote (−/−) is devoid of transcription of VMAT2 (top). G3PDH (983 bp) was used as internal control (bottom).
(C) Western blot analysis of whole brain synaptic vesicles. Samples (25 μg) of vesicles were solubilized and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 14%), transferred to nitrocellulose, subjected to Western blot analysis with anti-VMAT2-Ct (top) or anti–α-tubulin (bottom) antibodies, and developed with chemiluminescence. Molecular mass markers (kDa) are shown on the left. To confirm equal loading and transfer of proteins, the blots were stripped and reprobed with an antibody to α-tubulin. No difference in the level of α-tubulin immunoreactivity was observed between wild-type littermate and heterozygous VMAT2 knockout as determined by densitometric analysis using internal dilutional standards from wild-type littermate brain.
(D) Tetrabenazine (TBZ) binding to brain vesicle preparations. Newborn whole brains were used to obtain vesicle preparations. Increasing concentrations of TBZ were used to inhibit binding of the tritiated analog 3H-dihydrotetrabenazine (1 nM). Each point represents the mean of three binding experiments performed in triplicate with separate brain vesicle preparations. The homozygote binding parallels that of nonspecific binding. KD = 0.1 nM and Bmax = 12 fmol/mg tissue.
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5. Figure 3 Phenotype and Survival Rate of Homozygous VMAT2 Knockout Mice
(A) The homozygote phenotype. The homozygous mice are hypoactive and prone to hypothermia, and they exhibit severe growth retardation. The photograph shows two 10-day-old littermates. The homozygote (smaller pup) weighs <1/6 of that of the other pup.
(B) VMAT2 knockout mice survival. Two lines of VMAT2 knockout mice have been bred continuously for comparison. Of all homozygotes born, most die within a few days after birth. The longest life span was 12 days.
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6. Figure 4 Analyses of Monoamine Content and Synthesis in Newborn Mice
(A) HPLC analysis of total brain content of monoamines and metabolites. Newborn whole-brain homogenates were used for HPLC assessment of the levels of monoamines and metabolites. Results are presented as the mean ± SEM for 8–11 brains. Dopamine (DA), norepinephrine (NE), and serotonin (5-HT) levels were negligible in homozygotes, whereas the metabolite levels remain approximately the same (DOPAC and HVA) or elevated (5-HIAA), suggesting an increase in the rate of degradation (*p < 0.05 versus control; Student's t test).
(B) L-DOPA and 5-HTP accumulation in the brain after NSD Newborn mice were injected s.c. with L-aromatic acid decarboxylase inhibitor 3-hydroxybenzylhydrazine (NSD-1015) at a dose of 100 mg/kg. Ninety minutes later, whole-brain homogenates were prepared and HPLC-EC was performed. Results are presented as the mean ± SEM of 5–8 animals per group. L-DOPA and 5-HTP levels are doubled in the homozygotes after NSD-1015 treatment, indicating an increase in the rate of synthesis.
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7. Figure 5 Dopamine Efflux Measurement by Cyclic Voltammetry
A 30 Hz, 90 pulse electrical stimulation was used to evoke dopamine release from newborn striatal slices. Data points were collected every 100 ms. Insets are cyclic voltammograms. In the wild-type littermate (+/+) slices, dopamine is detected. There was no detectable dopamine release from the homozygote (−/−) slices.
Neuron  , DOI: ( /S (00) )

8. Figure 6
Impaired Striatal Dopaminergic Transmission in Adult VMAT2 Heterozygous Mice
(A) Tissue levels of monoamines and their metabolites in the striatum of adult VMAT2 heterozygote (+/−) and wild-type littermate (+/+) mice measured by HPLC-EC. Results are presented as means ± SEM of six mice per group of both VMAT2+/− and VMAT2+/+ mice (*p < 0.05 versus wild-type littermates; Student's t test).
(B) Extracellular dopamine levels in the striatum of freely moving mice measured using quantitative low perfusion rate microdialysis approach (Smith et al. 1992). Means ± SEM are shown (n = 8 for VMAT2+/− and 5 for VMAT2+/+ mice; *p < 0.05 versus wild-type littermates, Student's t test).
(C) Effect of high K+ (100 mM) depolarization on the extracellular dopamine level in the striatum of freely moving mice assessed by
microdialysis. Data are presented as a percentage of the average value of dopamine measured in at least three samples collected before the drug administration. Data points are means ± SEM (n = 8 for VMAT2+/− and 4 for VMAT2+/+ mice).
(D) Effect of amphetamine (10 mg/kg. i.p.) on the extracellular dopamine level in the striatum of freely moving mice. Data are presented as a percentage of the average value of dopamine measured in at least three samples collected before the drug administration. Results are presented as means ± SEM (n = 7 for VMAT2+/− and 6 for VMAT2+/+ mice).
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9. Figure 7
Effects of Apomorphine, Cocaine, Amphetamine, and Ethanol on the Locomotor Activity of VMAT2 Heterozygous and Wild-Type Littermates
(A) Time course of climbing behavior, measured as the time spent by the animal in the vertical position per each 5 min interval for VMAT2 heterozygous (+/−) and wild-type littermates (+/+) after apomorphine (2 mg/kg s.c.) treatment. Data points indicate means ± SEM.
(B–D) Time course of horizontal locomotor response of VMAT2+/− and VMAT2+/+ mice in response to cocaine ([B]; 20 mg/kg i.p.), to amphetamine ([C]; 0.5 mg/kg i.p.), and to ethanol ([D] 2 g/kg i.p.). Data are presented as means ± SEM.
Neuron  , DOI: ( /S (00) )

10. Figure 8
Locomotor Activity Responses in VMAT2 Wild-Type Littermate and Heterozygous Mice Subjected to a Chronic Cocaine Sensitization Paradigm
The mice were injected daily with cocaine (20 mg/kg i.p.) for 6 days, and 48 hr after the last injection animals were challenged with the same dose of the drug. Locomotor activity measurements were performed on days 1 and 8. The animals were habituated to the locomotor activity monitor for 1 hr before the drug administration. The activating effect of cocaine on locomotion is presented for the first 15 min after administration, where concomitant stereotyped behaviors characteristic for this dose of cocaine (Hiroi et al. 1997) were negligible. Analysis of accumulated distances traveled by the mice over 30 min, 1 hr, and 2 hr also revealed a lack of further sensitization in VMAT2 heterozygous (+/−) mice (data not shown). Data are presented as means ± SEM (**p < 0.01; ***p < versus wild-type littermates for the first day group; Student's t test).
Neuron  , DOI: ( /S (00) )