A minigene is a minimal gene fragment that includes an

exon An exon is any part of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term ''exon'' refers to both the DNA sequence within a gene and to the corresponding sequen ...

and the control regions necessary for the gene to express itself in the same way as a

wild type The wild type (WT) is the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "m ...

gene fragment. This is a minigene in its most basic sense. More complex minigenes can be constructed containing multiple exons and intron(s). Minigenes provide a valuable tool for researchers evaluating splicing patterns both ''

in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...

'' and ''

in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...

'' biochemically assessed experiments. Specifically, minigenes are used as splice reporter vectors (also called exon-trapping vectors) and act as a probe to determine which factors are important in splicing outcomes. They can be constructed to test the way both

cis-regulatory element ''Cis''-regulatory elements (CREs) or ''Cis''-regulatory modules (CRMs) are regions of non-coding DNA which regulate the transcription of neighboring genes. CREs are vital components of genetic regulatory networks, which in turn control morphoge ...

s (RNA effects) and

trans-regulatory element Trans-regulatory elements (TRE) are DNA sequences encoding upstream regulators (ie. trans-acting factors), which may modify or Regulation of gene expression, regulate the expression of distant genes. Trans-acting factors interact with Cis-regulatory ...

s (associated

proteins Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

splicing factor A splicing factor is a protein involved in the removal of introns from strings of messenger RNA, so that the exons can bind together; the process takes place in particles known as spliceosomes. Genes are progressively switched off as we age, and sp ...

s) affect gene expression. Splice2

History

Minigenes were first described as the somatic assembly of DNA segments and consisted of DNA regions known to encode the protein and the flanking regions required to express the protein. The term was first used in a paper in 1977 to describe the cloning of two minigenes that were designed to express a peptide. RNA splicing was discovered in the late 1970s through the study of adenoviruses that invade mammals and replicate inside them. Researchers identified RNA molecules that contained sequences from noncontiguous parts of the virus’s genome. This discovery led to the conclusion that regulatory mechanisms existed which affected mature RNA and the genes it expresses. Using minigenes as a splice reporting vector to explore the effects of RNA splicing regulation naturally followed and remains the major use of minigenes to date.

Types

In order to provide a good minigene model, the gene fragment should have all of the necessary elements to ensure it exhibits the same alternative splicing (AS) patterns as the

gene, i.e., the length of the fragment must include all upstream and downstream sequences which can affect its splicing. Therefore, most minigene designs begin with a thorough ''

in silico In biology and other experimental sciences, an ''in silico'' experiment is one performed on computer or via computer simulation. The phrase is pseudo-Latin for 'in silicon' (correct la, in silicio), referring to silicon in computer chips. It ...

'' analysis of the requirements of the experiment before any "wet" lab work is conducted. With the advent of

Bioinformatics Bioinformatics () is an interdisciplinary field that develops methods and software tools for understanding biological data, in particular when the data sets are large and complex. As an interdisciplinary field of science, bioinformatics combi ...

and widespread use of computers, several good programs now exist for the identification of cis-acting control regions that affect the splicing outcomes of a gene and advanced programs can even consider splicing outcomes in various tissue types. Differences in minigenes are usually reflected in the final size of the fragment, which is in turn a reflection of the complexity of the minigene itself. The number of foreign DNA elements (exon and introns) inserted into the constitutive exons and introns of a given fragment varies with the type of experiment and the information being sought. A typical experiment might involve

minigenes which are expected to express genes normally in a comparison run against genetically engineered allelic variations which replace the wild-type gene and have been cloned into the same flanking sequences as the original fragment. These types of experiments help to determine the effect of various mutations on

pre-mRNA A primary transcript is the single-stranded ribonucleic acid (RNA) product synthesized by transcription of DNA, and processed to yield various mature RNA products such as mRNAs, tRNAs, and rRNAs. The primary transcripts designated to be mRNAs a ...

splicing.

Construction

Once a suitable genomic fragment is chosen (Step 1), the exons and introns of the fragment can be inserted and amplified, along with the flanking constitutive exons and introns of the original gene, by PCR. The primers for PCR can be chosen so that they leave "

sticky ends DNA ends refer to the properties of the ends of linear DNA molecules, which in molecular biology are described as "sticky" or "blunt" based on the shape of the complementary strands at the terminus. In sticky ends, one strand is longer than the ...

" at 3' sense and anti-sense strands (Step 2). These "sticky-ends" can be easily incorporated into a TOPO Vector by ligation into a commercially available source which has ligase already attached to it at the sight of incorporation (Step 3). The subsequent TOPO Vectors can be transfected into E.coli cells (Step 4). After incubation, total RNA can be extracted from the bacterial colonies and analyzed using

RT-PCR Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase cha ...

to quantify ratios of exon inclusion/exclusion (step 5). The minigene can be transfected into different cell types with various splicing factors to test trans-acting elements (Step 6). The expressed genes or the proteins they encode can be analyzed to evaluate splicing components and their effects via a variety of methods including

hybridization Hybridization (or hybridisation) may refer to: *Hybridization (biology), the process of combining different varieties of organisms to create a hybrid *Orbital hybridization, in chemistry, the mixing of atomic orbitals into new hybrid orbitals *Nu ...

size-exclusion chromatography Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules ...

Uses

RNA splicing RNA splicing is a process in molecular biology where a newly-made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). It works by removing all the introns (non-coding regions of RNA) and ''splicing'' b ...

errors have been estimated to occur in a third of genetic diseases. To understand

pathogenesis Pathogenesis is the process by which a disease or disorder develops. It can include factors which contribute not only to the onset of the disease or disorder, but also to its progression and maintenance. The word comes from Greek πάθος ''pat ...

and identify potential targets of therapeutic intervention in these diseases, explicating the splicing elements involved is essential. Determining the complete set of components involved in splicing presents many challenges due to the abundance of alternative splicing, which occurs in most human genes, and the specificity in which splicing is carried out ''in vivo''. Splicing is distinctly conducted from cell type to cell type and across different stages of cellular development. Therefore, it is critical that any ''

'' or bioinformatic assumptions about splicing regulation are confirmed ''in vivo''. Minigenes are used to elucidate ''cis''-regulatory elements, ''trans''-regulatory elements and other regulators of pre-mature RNA splicing ''in vivo''. Minigenes have been applied to the study of a diverse array of genetic diseases due to the aforementioned abundance of alternatively spliced genes and the specificity and variation observed in splicing regulation. The following are examples of minigene use in various diseases. While it is not an exhaustive list, it does provide a better understanding of how minigenes are utilized.

Endocrine diseases

RNA splicing errors can have drastic effects on how proteins function, including the hormones secreted by the

endocrine system The endocrine system is a messenger system comprising feedback loops of the hormones released by internal glands of an organism directly into the circulatory system, regulating distant target organs. In vertebrates, the hypothalamus is the neu ...

. These effects on hormones have been identified as the cause of many endocrine disorders including thyroid-related pathological conditions,

rickets Rickets is a condition that results in weak or soft bones in children, and is caused by either dietary deficiency or genetic causes. Symptoms include bowed legs, stunted growth, bone pain, large forehead, and trouble sleeping. Complications may ...

hyperinsulinemic hypoglycemia Hyperinsulinemic hypoglycemia describes the condition and effects of low blood glucose caused by excessive insulin. Hypoglycemia due to excess insulin is the most common type of serious hypoglycemia. It can be due to endogenous or injected insulin ...

and

congenital adrenal hyperplasia Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders characterized by impaired cortisol synthesis. It results from the deficiency of one of the five enzymes required for the synthesis of cortisol in the adrenal cortex. ...

. One specific example of a splicing error causing an endocrine disease that has been studied using minigenes is a type of

growth hormone deficiency Growth hormone deficiency (GHD), or human growth hormone deficiency, is a medical condition resulting from not enough growth hormone (GH). Generally the most noticeable symptom is that an individual attains a short height. Newborns may also presen ...

called isolated growth hormone deficiency (IGHD), a disease that results in growth failure. IGHD type II is an autosomal dominant form caused by a mutation in the intervening sequence (IVS) adjacent to exon 3 of the gene encoding growth hormone 1, the GH-1 gene. This mutated form of IVS3 causes exon 3 to be skipped in the mRNA product. The mRNA (-E3) encodes a truncated form of hGH that then inhibits normal hGH secretion. Minigenes were used to determine that a

point mutation A point mutation is a genetic mutation where a single nucleotide base is changed, inserted or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product—consequences ...

within an intron splice enhancer (ISE) embedded in IVS3 was to blame for the skipping of E3. Moreover, it was determined that the function of the ISE is influenced by a nearby transposable AC element, revealing that this particular splicing error is caused by a trans-acting factor.

Neurodegenerative diseases

Accumulation of

tau protein The tau proteins (abbreviated from tubulin associated unit) are a group of six highly soluble protein isoforms produced by alternative splicing from the gene ''MAPT'' (microtubule-associated protein tau). They have roles primarily in maintaining ...

is associated with

neurodegenerative diseases A neurodegenerative disease is caused by the progressive loss of structure or function of neurons, in the process known as neurodegeneration. Such neuronal damage may ultimately involve cell death. Neurodegenerative diseases include amyotrophic ...

including

Alzheimer's Alzheimer's disease (AD) is a neurodegenerative disease that usually starts slowly and progressively worsens. It is the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in remembering recent events. As t ...

and

Parkinson's Parkinson's disease (PD), or simply Parkinson's, is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. The symptoms usually emerge slowly, and as the disease worsens, non-motor symptoms becom ...

diseases as well as other

tauopathies Tauopathy belongs to a class of neurodegenerative diseases involving the aggregation of tau protein into neurofibrillary or gliofibrillary tangles in the human brain. Tangles are formed by hyperphosphorylation of the microtubule protein known a ...

. Tau protein isoforms are created by alternative splicing of exons 2, 3 and 10. The regulation of tau splicing is specific to stage of development, physiology and location. Errors in tau splicing can occur in both exons and introns and, depending on the error, result in changes to protein structure or loss of function. Aggregation of these abnormal tau proteins correlates directly with pathogenesis and disease progression. Minigenes have been used by several researchers to help understand the regulatory components responsible for mRNA splicing of the TAU gene.

Cancer

Cancer is a complex, heterogeneous disease that can be hereditary or the result of environmental stimuli. Minigenes are used to help oncologists understand the roles

splicing plays in different cancer types. Of particular interest are cancer specific genetic mutations that disrupt normal splicing events, including those affecting

spliceosome A spliceosome is a large ribonucleoprotein (RNP) complex found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs (snRNA) and numerous proteins. Small nuclear RNA (snRNA) molecules bind to specifi ...

components and

RNA-binding protein RNA-binding proteins (often abbreviated as RBPs) are proteins that bind to the double or single stranded RNA in cells and participate in forming ribonucleoprotein complexes. RBPs contain various structural motifs, such as RNA recognition motif ( ...

s such as heterogeneous nuclear ribonucleoparticules (hnRNP), serine/arginine-rich (SR) proteins and small ribonucleoproteins (snRNP). Proteins encoded by aberrantly spliced pre-mRNAs are functionally different and contribute to the characteristic anomalies exhibited by cancer cells, including their ability to proliferate, invade and undergo angiogenesis, and metastasis. Minigenes help researchers identify genetic mutations in cancer that result in splicing errors and determine the downstream effects those splicing errors have on gene expression. Using knowledge obtained from studies employing minigenes, oncologists have proposed tests designed to detect products of abnormal gene expression for diagnostic purposes. Additionally, the prospect of using minigenes as a

cancer immunotherapy Cancer immunotherapy (sometimes called immuno-oncology) is the stimulation of the immune system to treat cancer, improving on the immune system's natural ability to fight the disease. It is an application of the fundamental research of cancer im ...

is being explored.

References

External links

*
Stefan Stamm's web page at the University of Kentucky
Good overview of minigene research.

A Good site for theoretical analysis of splicing.
UCSC Genome Browser
Large database for retrieving information on genes. Molecular biology * Biotechnology Synthetic biology Molecular genetics Cloning