Knockout or Knock-in? A Truncated D2 Receptor Protein Is Expressed in the Brain of Functional D2 Receptor Knockout Mice
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. Horizontal Locomotor Activity
2.3. Bioinformatics Research
2.4. RT-PCR and 3′RACE
2.5. Cloning of Truncated D2R and Immunodetection in the HEK293T Cell Line
2.6. In Vivo Brain Microdialysis
2.7. Analysis of Dialysate Samples
2.8. Immunofluorescence in Brain Tissue
2.9. Statistical Analysis
3. Results and Discussion
3.1. D2R Functional Knockout Mice Express Mature mRNA Transcripts That Lack Only Exon Eight
3.2. D2R Functional Knockout Mouse Brain Bears a Protein Recognizable by Anti-D2R Antibodies
3.3. Recombinant Truncated D2R Shows Less Localization in the Golgi Apparatus Than Native D2R in HEK293T Cell Line
3.4. Diminished Locomotor Activity and Extracellular Dopamine Levels in D2R Functional Knockout Mice
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Method | Primer | Sequence (5′–3′) |
---|---|---|
Genotyping | P8036 | TGATGACTGGGAATGTTGGTGTGC |
P8037 | CTCCCCAGACTTGTGGCAAAAGG | |
P8038 | AGGATTGGGAAGACAATAGCAG | |
RT-PCR | D2 Fw | CCCTTCATCGTCACCCTGCTGG |
D2 Rv | CTCCATTTCCAGCTCCTGAG | |
RT-PCR polyA transcripts | D2—1(Fw) | CTCAGGAGCTGGAAATGGAG |
Transcription (poly-T) | GCTCAAGCTTTTTTTTTTTTTTTTT | |
3′RACE | QT (Rv) | CCAGTGAGCAGAGTGACGAGGACTCGA |
D2—1(Fw) nested PCR1 | CTCAGGAGCTGGAAATGGAG | |
D2—2(Fw) nested PCR2 | CCACGGTCTACATAGCAACC | |
Q0 (Rv) nested PCR1 | CCAGTGAGCAGAGTGACG | |
Q1 (Rv) nested PCR2 | GAGGACTCGAGCTCAAGC | |
D2R subcloning | EcoRI-ATG-Flag-D2R Fw | GCATGAATTCATGGACTACAAGGACGATGATGACGCC |
Flag-D2R-XhoI Rv | GCCTTCCTGAAGATCCTCCACTGCTGACTCGAGGCAT | |
FLAG-D2R-ΔE8 Rv | ATGCCTCGAGTCAGAGAACAATGGCGAGCATC |
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Sánchez, N.; Olivares-Costa, M.; González, M.P.; Munita, R.; Escobar, A.P.; Meza, R.; Herrera-Rojas, M.; Albornoz, J.; Merello, G.; Andrés, M.E. Knockout or Knock-in? A Truncated D2 Receptor Protein Is Expressed in the Brain of Functional D2 Receptor Knockout Mice. NeuroSci 2021, 2, 193-206. https://doi.org/10.3390/neurosci2020014
Sánchez N, Olivares-Costa M, González MP, Munita R, Escobar AP, Meza R, Herrera-Rojas M, Albornoz J, Merello G, Andrés ME. Knockout or Knock-in? A Truncated D2 Receptor Protein Is Expressed in the Brain of Functional D2 Receptor Knockout Mice. NeuroSci. 2021; 2(2):193-206. https://doi.org/10.3390/neurosci2020014
Chicago/Turabian StyleSánchez, Natalia, Montserrat Olivares-Costa, Marcela P González, Roberto Munita, Angélica P Escobar, Rodrigo Meza, Mauricio Herrera-Rojas, Jessica Albornoz, Gianluca Merello, and María E Andrés. 2021. "Knockout or Knock-in? A Truncated D2 Receptor Protein Is Expressed in the Brain of Functional D2 Receptor Knockout Mice" NeuroSci 2, no. 2: 193-206. https://doi.org/10.3390/neurosci2020014