Minigenes

Laura E Warner, University of Washington, Seattle, Washington, USA
Jeffrey S Chamberlain, University of Washington, Seattle, Washington, USA

Published online: January 2006

DOI: 10.1038/npg.els.0005684

A minigene is a compact version of a gene in which regions have been removed without affecting protein function. Understanding the structure and function of the protein produced from the gene of interest is critical in determining which portions of the gene can be deleted. The compact size of minigenes makes them easier to isolate and insert into cloning and viral vectors.

Keywords: DMD; dystrophin; factor VIII; hemophilia; gene therapy

Figure 1. Creating a minigene. This diagram shows how a large gene can be converted into a compact minigene. The genomic structure of gene X is shown. This gene is translated into mRNA and then converted into cDNA. The nonessential coding exons and untranslated regions (UTRs) are removed and essential regulatory elements are added. P1: gene X promoter; E: enhancer of gene X promoter; pA-1: polyA site for gene X; P2: generic promoter; pA-2: generic polyA site.
Figure 2. Construction of dystrophin minigenes for placement in viruses by determining the essential structural domains. (a) At the top is a representation of the full-length dystrophin protein showing its four major structural domains: N-terminal actin-binding domain (ABD); the rod domain composed of four hinges (boxes numbered H1–H4) and 24 repeated units (boxes numbered R1–R24); the cysteine-rich domain (CR); and C-terminal domain (CT). Below are the dystrophin deletion constructs used to determine the functional importance of these structural domains by evaluating their ability to prevent dystrophy. Using this information, dystrophin minigenes have been constructed that are capable of fitting into adenoassociated virus (AAV) (b) or adenovirus (c). The numbers indicate the size of each minigene construct.
Figure 3. Construction of factor VIII (FVIII) minigenes by deletion of nonessential regions. (a) Domain structure and processing of FVIII. The structural domains are shown: A1–A3, B and C1–C2. The dotted boxes are regions that are rich in acidic amino acid residues and contain sites of tyrosine sulfation. The asterisk indicates the residue (1680) essential for binding von Willebrand factor (vWF). Arrows indicate the position of the essential cleavage sites involved in FVIII processing. FVIII minigenes have been placed into adenovirus (b) or adenoassociated virus (AAV) (c). The numbers indicate the size of each minigene construct. (Modified from Kaufman RJ (1999) Advances toward gene therapy for hemophilia at the millennium. Human Gene Therapy 10: 2091–2107.)
close
 References
    book Amalfitano A, Rafael JA and Chamberlain JS (1997) "Structure and mutation of the dystrophin gene". In: Lucy JA and Brown SC (eds.) Dystrophin: Gene, Protein and Cell Biology, pp. 1–26. Cambridge, UK: Cambridge University Press.
    Connelly S, Smith TA, Dhir G, et al. (1995) In vivo gene delivery and expression of physiological levels of functional human factor VIII in mice. Human Gene Therapy 6: 185–193.
    Corrado K, Rafael JA, Mills PL, et al. (1996) Transgenic mdx mice expressing dystrophin with a deletion in the actin-binding domain display a ‘mild Becker’ phenotype. Journal of Cell Biology 134: 873–884.
    Crawford GE, Faulkner JA, Crosbie RH, et al. (2000) Assembly of the dystrophin-associated protein complex does not require the dystrophin COOH-terminal domain. Journal of Cell Biology 150: 1399–1410.
    Gnatenko DV, Saenko EL, Jesty J, et al. (1999) Human factor VIII can be packaged and functionally expressed in an adeno-associated virus background: applicability to haemophilia A gene therapy. British Journal of Haematology 104: 27–36.
    Hartigan-O'Connor D and Chamberlain JS (2000) Developments in gene therapy for muscular dystrophy. Microscopy Research and Technique 48: 223–238.
    Kaufman RJ (1999) Advances toward gene therapy for hemophilia at the millennium. Human Gene Therapy 10: 2091–2107.
    Rafael JA, Cox GA, Corrado K, et al. (1996) Forced expression of dystrophin deletion constructs reveals structure–function correlations. Journal of Cell Biology 134: 93–102.
    Straub V and Campbell KP (1997) Muscular dystrophies and the dystrophin–glycoprotein complex. Current Opinion in Neurology 10: 168–175.
    Toole JJ, Pittman DD, Orr EC, et al. (1986) A large region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity. Proceedings of the National Academy of Sciences of the United States of America 83: 5939–5942.
 Further Reading
    Deconinck N, Ragot T, Maréchal G, Perricaudet M and Gillis JM (1996) Functional protection of dystrophic mouse (mdx) muscles after adenovirus-mediated transfer of a dystrophin minigene. Proceedings of the National Academy of Sciences of the United States of America 93: 3570–3574.
    Eaton D, Rodriguez H and Vehar GA (1986) Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry 25: 505–512.
    book Emery AEH (1993) Duchenne Muscular Dystrophy. Oxford, UK: Oxford Medical Publications.
    Emilien G, Maloteaux JM, Penasse C, Goodeve A and Casimir C (2000) Haemophilias: advances towards genetic engineering replacement therapy. Clinical and Laboratory Haematology 22: 313–323.
    England SB, Nicholson LV, Johnson MA, et al. (1990) Very mild muscular dystrophy associated with the deletion of 46% of dystrophin. Nature 343: 180–182.
    Koenig M, Monaco AP and Kunkel LM (1988) The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 53: 219–226.
    Leyte A, van Schijndel HB, Niehrs C, et al. (1991) Sulfation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor. Journal of Biological Chemistry 266: 740–746.
    Monaco AP, Bertelson CJ, Liechti-Gallati S, Moser H and Kunkel LM (1988) An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 2: 90–95.
    Toole JJ, Knopf JL, Wozney JM, et al. (1984) Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 312: 342–347.
    book Waston J, Gilman M, Witkowski J and Zoller M (1992) Recombinant DNA. New York, NY: WH Freeman and Company.
 Web Links
    ePath Coagulation factor VIII, procoagulant component (hemophilia A) (F8); Locus ID: 2157. LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=2157
    ePath Dystrophin (muscular dystrophy, Duchenne and Becker types) (DMD); Locus ID: 1756. LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=1756
    ePath Coagulation factor VIII, procoagulant component (hemophilia A) (F8); MIM number: 306700. OMIM: http://www.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?306700
    ePath Dystrophin (muscular dystrophy, Duchenne and Becker types) (DMD); MIM number: 300377. OMIM: http://www.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?300377

Related Sub-Topics:

Translation and Protein synthesis | Transcription | Sample Preparation in Structural Biology | Gene and Genome Structure and Organisation | Gene Therapy | DNA Structure and Function | Techniques and Tools in Molecular Biology | Evolution of Coding and Functional Modules
Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

Article Title: Minigenes
 
How to Cite close
Warner, Laura E, and Chamberlain, Jeffrey S(Jan 2006) Minigenes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0005684]