This document summarizes a proposed experiment to increase RELN transcription in reeler mice by delivering the Tbr1 transcription activator using exosome treatment. The experiment involves isolating the Tbr1 gene from healthy mouse brain tissue, amplifying it via PCR, recombining it into a pUC19 plasmid, transforming E. coli with the plasmid to produce purified Tbr1 protein, and injecting the protein into exosomes derived from dendritic cells. The exosomes would then be administered to cultured reeler mouse brain tissue and to reeler mice via nasal spray to potentially increase RELN expression, improve neural connectivity, and alleviate schizophrenic symptoms. The results would be analyzed using microarrays and behavioral observations.

1. Treating Schizophrenia: Keepin’ it Reelin
Bradley Van Rooy, Jessica Dewey, Tracy Dinh, Kate Geschwind, Willie Meder, Colleen Smith, Jonah Widmer, Kristen Woodhouse
Schizophrenia: A Chronic Psychotic Syndrome. 1,2
•Patients falsely interpret reality
•Severely debilitating symptoms include delusions, hallucinations,
and impaired social and communicative functioning
•2.4 million patients in America each year
May be caused by decreased expression of RELN gene 3,4,5
•RELN protein product, reelin, causes appropriate neuronal positioning
•50% less reelin in schizophrenic patients
•Due to hypermethylation of RELN gene
RELN Transcription Factor: T-box brain 1 is the protein product of Tbr16,7,8
•Less Tbr1 expression means less reelin
•More Tbr1 means more reelin
Reeler mice (Mus musculus) as a model organism for schizophrenia9,10,11,12
•Deleterious RELN gene disrupts reelin signaling pathway
•Heterozygous mice have 50% less reelin
Our experiment seeks to increase RELN transcription in reeler mice
•Deliver transcription activator to mouse brains using exosome
treatment
•The goal is to increase RELN expression, improve neural connectivity,
and achieve therapeutic alleviation of schizophrenic symptoms
RELN: An ancestral
character for the
Eutheria clade.13
• Maximum parsimony
phylogenetic tree shows
evolutionary relationship
between humans, mice
• Orthologs of Homo sapiens
RELN have been located in 86
mammals.
• H. sapiens RELN is conserved in
9 of these mammals, including
Mus musculus.14
• Research methods applied in
M. musculus may eventually be
applied to orthologous species.
• Large number of RELN paralogs
exist in every species that
carries RELN.
pUC19 Plasmid contains all
necessary coding regions. 21,34
• rep region causes replication.
• bla region (Apr) confers ampicillin resistance as screening system.
• lacZ gene, its promoter, lac repressor binding site as reporter system.
• Tbr1 inserted at multiple cloning site.
Animal research must be ethical. 35
•Clear purpose of benefit and justified as a “last resort” measure
•Animal welfare maintained
•Follow IACUC guidelines for animal treatment as per www.iacuc.org
•Humane surgical procedures; multiple surgeries approved
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phylogeny using phylogenomics and the multispecies coalescent model. PNAS, 109(37), 14942-14947. Retrieved from http://www.pnas.org/content/109/37/14942.long, 14. RELN reelin [ homo sapiens (human) ]. (2014, April 21). Retrieved from http://www.ncbi.nlm.nih.gov/gene/5649, 15. Rice, G. DNA Extraction. Montana State University, Microbial Life Educational Resources. Carleton.edu 14 December 2013, 16. Mortlock, D., Nelson, M., Innis, J., 2014. An efficient method for isolating putative promoters and 5’ -transcribed sequences from large genomic clones. Genome Research 1996, 327, 17. The immunogenicity
of dendritic cell-derived exosomes. Quah BJ1, O'Neill HC. Blood Cells Mol Dis. 2005 Sep-Oct;35(2):94-110. Accessed at http://www.ncbi.nlm.nih.gov/pubmed/15975838., 18. Purification, Characterization and Biological Significance of Tumor-derived Exosomes. Kenichiro Koga, et al. Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. ANTICANCER RESEARCH 25: 3703-3708 (2005). http://ar.iiarjournals.org/content/25/6A/3703.full.pdf, 19. Primers/Oligos, Cloning and Gene Synthesis, www.lifetechnologies.com, 20. PCR: Introduction. (n.d.). NCBI. Retrieved April 22, 2014, from
http://www.ncbi.nlm.nih.gov/genome/probe/doc/TechPCR.shtml, 21. Han, Y., Chen, L., Guan, L., He, S., 2014. Inverse PCR-Based method for isolating novel SINEs from genome. Molecular Biotechnology 56, 296–304. doi:10.1007/s12033-013-9708-y, 22. Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 7.1, DNA Cloning with Plasmid Vectors. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21498, 23. DNA ligation. (n.d.). Retrieved from https://www.addgene.org/plasmid_protocols/DNA_ligation/, 24. Nallamsetty, S., Waugh, D., 2007. A generic
protocol for the expression and purification of recombinant proteins in Escheria coli using a combinatorial His6-matose binding protein fusion tag. Nature Protocols 2, 383–391. doi:10.1038/nprot.2007.50, 25. Cabrita, L., Dai, W., Bottomley, S., 2006. A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production. BMC Biotechnol.6, 12. doi:10.1186/1472-6750-6-12, 26. Hartley, J., Temple, G., Brasch, M., 2000. DNA cloning using in vitro site-specific recombination. Genome Research 10, 1788–1795., 27. Griffiths AJF, Gelbart WM, Miller JH, et al. Modern Genetic Analysis. New
York: W. H.Freeman; 1999. Bacterial Transformation. 28. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Alvarez-Erviti L1, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ. Nat Biotechnol. 2011 Apr;29(4):341-5. doi: 10.1038/nbt.1807. Epub 2011 Mar 20. http://www.ncbi.nlm.nih.gov/pubmed/21423189, 29. NIH awards University of Chicago $1.5 million to study novel therapy for multiple sclerosis. Kevin Jiang. UChicagoNews. SEPTEMBER 18, 2013.http://news.uchicago.edu/article/2013/09/18/nih-awards-university-chicago-15-million-study-novel-therapy-multiple-sclerosis, 30. Soverchia, L.,
Ubaldi, M., Leonardi‐Essmann, F., Ciccocioppo, R., &Hardiman, G. (2005).Microarrays‐The Challenge of Preparing Brain Tissue Samples. Addiction biology, 10(1), 5-13. 31. Hegde, P., Qi, R., Abernathy, K., Gay, C., Dharap, S., Gaspard, R., ... &Quackenbush, J. (2000).A concise guide to cDNA microarray analysis. Biotechniques, 29(3), 548-563. 32. Matsuki, T., Hori, G., &Furuichi, T. (2005).Gene expression profiling during the embryonic development of mouse brain using an oligonucleotide-based microarray system. Molecular brain research, 136(1), 231-254. 33. Sato, N., Fukushima, N., Chang, R., Matsubayashi, H., &Goggins,
M. (2006). Differential and Epigenetic Gene Expression Profiling Identifies Frequent Disruption of the RELN Pathway in Pancreatic Cancers. Gastroenterology, 130(2), 548-565, 34. pUC19 vector. (n.d.). Retrieved from http://www2.le.ac.uk/departments/genetics/genie/images/project-related-images/pUC19.jpg/view, 35. American Psychological Association. (2012, February 24).Guidelines for ethical conduct in the care and use of nonhuman animals in research. Retrieved from https://www.apa.org/science/leadership/care/guidelines.aspx?item=1, 36. "Trials of War Criminals before the Nuremberg Military Tribunals under
Control Council Law No. 10", Vol. 2, pp. 181-182. Washington, D.C.: U.S. Government Printing Office, 1949. Accessed at http://www.hhs.gov/ohrp/archive/nurcode.html , 37. Department of Health, Education, and Welfare. The National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research Sess. (1979) Washington, D.C. IMAGE CREDITS: Experimental design images retrieved from: http://pegstookey.legacycentered.com/files/2013/03/Brain-clipart.png, http://pixabay.com/p-296581/?no_redirect,
http://sweetclipart.com/multisite/sweetclipart/files/dna_black.png, http://upload.wikimedia.org/wikipedia/commons/0/07/Protein_HBZ_PDB_1jeb.png, http://www.clker.com/cliparts/B/o/K/L/E/9/cho-cell-petri-dish2-hi.png, http://www.ipharmd.net/images/blue_filled_syringe.png, http://anniehalliday.com/biopics/composite/microarray.jpg. Phylogenetic Tree adapted from http://www2.le.ac.uk/departments/genetics/genie/images/project-related-images/pUC19.jpg/image_preview. Plasmid image adapted from http://filebox.vt.edu/users/chagedor/biol_4684/Methods/transformed.gif. Tbr1 gene image adapted
from http://www.ncbi.nlm.nih.gov/gene/10716.
Future research may investigate
alternative human therapies.
•Alternative genes and gene products
•Alternative brain regions
•Environmental triggers
•Extension of research towards effects of RELN gene
•Differentiation of neurons
•Neurogenesis in the hippocampus
•Effects on other neuropathologies
•Additional research on animals more closely related to humans (pigs, monkeys, etc.)
•Eventual research with human patients36,37
•Informed consent and required ability to stop research
•Avoid unnecessary harm and maximize benefits
Will increasing RELN transcription
have a therapeutic benefit?
•Poor neuronal orientation is only one aspect of complex disease pathology
•Possible procedural errors:
•Isolation of Tbr1, amplification during PCR, plasmid
recombination, or transformation of E. coli
•DCDE may not be suitable carrier for Tbox brain 1 due to
unforeseen size or other molecular qualities
•Problematic brain tissue culturing or removal
•Failure to target correct RELN paralog may complicate schizophrenic pathology
Tissue Collection & Exosome Isolation
• Isolate DNA from brain tissue of healthy mouse to obtain functional
Tbr1 gene15,16
• Surgically remove and cultivate brain tissue cultures from reeler
mice17
• Extract self-derived dendrite cell-derived exosomes through
centrifugation18
PCR Amplification & Plasmid Recombination
• Amplify Tbr1 gene through PCR using two custom-designed
primers: EcoRI/BamHI binding site + complementary 3’ DNA19,20,21,22
•Insert Tbr1 gene into E. coli plasmid pUC19 using restriction
enzymes EcoRI and BamHI23
E. coli Transformation with pUC1923,24,25,26
• Culture E. coli in appropriate media27
•Test for Tbr1 using complementary cDNA probe
•Collect and purify Tbr1 protein sample
In Vitro Exosome Treatment
• Experimental group: inject purified Tbr1 protein into exosomes and
add to cultured brain tissue28
• Control group: inject fluorescent marker protein into exosomes and
add to cultured brain tissue28
• Analyze differential transcription of in vitro cells with microarray.
• Proceed to in situ experiment if control group protein is detected
within neurons and RELN expression increased in experimental
group
In Situ Exosome Treatment
• Experimental group: inject purified protein into self-derived
exosomes and administer nasally via spray29
• Control group: inject fluorescent marker protein into self-derived
exosomes and administer nasally via spray29
• Repeat exosome administration daily for several weeks
Microarray and Behavioral Analysis
• Record observations of mice behaviors and schizophrenia-
resembling symptoms
• Analyze relative RELN transcription using microarray30,31,32,33
Experimental Design
References
BIOL 2002 – Section 1; Team 10; Spring 2014
Tbr1 Gene
11,412 bp