Top 10 Model Organisms

Conducting trials directly on humans is forbidden on humanitarian grounds. Since the human body is very complex direct involvement of humans as subjects of experiments may not always be possible. Therefore it is ideal to use prokaryotes, eukaryotes, and organisms other than humans. These helper organisms are known as model organisms. They contribute to scientific and industrial research. These organisms include bacteria, fungi, viruses, algae, nematodes, worms, and higher animals. Model organisms are selected based on criteria such as their ease of growing, short life cycle, well-defined cellular structures, easily analyzable properties, safe and ethically sound. Using hazardous toxin-producing bacteria or organisms for conducting experiments is forbidden and is accessed only in rare and unavoidable cases. Only trained, well-qualified professionals handle these organisms. Model organisms are involved in genome analysis, recombination experiments and sequence similarity studies. It requires a lot of care and caution to handle these organisms. Recombinant vaccines, peptides, and biomolecules are produced using model organisms. Biosatellites and spaceships use model organisms in their first launch.

1.   Yeast:

Yeast is a very useful eukaryote with beneficial properties. Yeast is very easy to culture because it can easily grow on fruits, sugary and salty foods. It can easily proliferate from 12⁰C to 40⁰C. The environment of acidic to alkaline is suitable for its growth. Beer and wine industry widely make use of yeast. Preparation of bakery products also involves yeast. Division of yeast occurs due to a process is known as budding. Yeast cells are easy to study since yeast is a single-celled organism. In addition to studying yeast cells with ease, these cells are comparatively safe to handle.

Most of the experiments in genetic engineering and genome research involve the use of yeast as a model organism. It is possible to study cell cycle, DNA replication, protein and cell wall synthesis using yeast cells. Gene cloning can be achieved using vectors. Yeast artificial chromosomes (YACs) are yeast cloning vectors. Typical features of YACs include telomere (TEL), centromere (CEN), selectable markers on each arm, an origin of replication, and restriction sites or multiple cloning sites. YAC vectors can accommodate DNA fragments of several base pairs. Protein-protein interaction studies make use of the yeast two-hybrid system. Cancer genetic studies also involve yeast as model organisms.

2.   Fruit fly:

Most of the genetics experiments involve the use of the fruit fly. It is scientifically known as Drosophila melanogaster. Drosophila is easy to grow since it does not require any specific conditions to grow. It can grow on a wide range of foodstuffs right from a piece of banana to a culture medium. There is a wide range of mutants observed in the fruit fly. Studying its genes is simpler as compared to other organisms. Drosophila is used to study developmental genes, mutant genes, and chromosomes. Morgan was the first scientist to use this model organism. The generation time of fruit fly is rapid as it takes less than two weeks to grow. Transgenic fruit flies are those that consist of foreign gene(s) inserted into their genome.

Drosophila is one such genus of the fruit flies that contains over 900 described species. The genic balance theory was used to identify the concept of sex determination. This theory worked out primarily in Drosophila. According to this theory, sex determination is the ratio of sex chromosomes and polytene chromosomes. A polytene chromosome is a giant chromosome in Drosophila produced by the endomitotic process. It consists of many chromatids. DNA replication and heterochromatin studies are carried out using Drosophila polytene chromosomes. They are also involved in gene mapping and linkage studies. The fruit fly is a model system for understanding human biology and disease. Findings suggest that this model organism has homologs for 177 out of 289 genes involved in human cancers.

Image: Model organisms

3.   E.coli:

Escherichia coli are most widely used bacteria in the biotechnology industry. Genetic engineering principles make very high use of this bacteria for cloning and other experiments. The life cycle of E. coli is simple to understand. Hence they are easy to grow and safe to handle. These bacteria consist of extrachromosomal material known as plasmids. Genetic modifications are possible with this model organism. E.coli can be engineered to produce the desired product. For example, gene modification helps in synthesizing insulin. Plasmid cloning vectors include pUC 19 vectors that contain multiple cloning sites. Important applications in model organisms are as follows:

a.    Gene cloning experiments: Pure samples of cloned DNA are obtained using plasmid vectors. Some of the plasmids with multiple cloning sites enable different types of restriction enzymes to act on them.

b.   Bacteriophage studies: The viruses that infect the bacteria are known as bacteriophages. They follow lytic as well as the lysogenic cycle of infection.

c.     Gene mapping: E.coli bacteria undergo a process of conjugation for transferring genes from one bacterium to another. Thus, gene mapping is possible with the help of conjugation experiments.

d.    Study of DNA replication: Prokaryotic DNA replication study involves E. coli as a model organism.

e.     Identification of mutant phages: E. coli are used to identify mutant phages in site-directed mutagenesis.

f.       Bacterial Artificial Chromosomes (BACs): They are useful for cloning DNA fragments in E. coli. BACs consists of an origin of replication, multiple cloning sites, a selectable marker, and other features.

4.   Nematode C. elegans:

Caenorhabditis elegans is the widely used nematode in zoology. This nematode exhibits anatomical simplicity. It is ideal to carry out gene diversity and cell cycle studies with this organism. The life cycle of this nematode is just three days. Early embryonic genes are clear to study with this organism. It is capable of learning simple tasks. C elegans is used to study the genetic and molecular aspects of embryonic development, morphogenesis, nerve systems, aging, and behavior.

5.   Arabidopsis thaliana:

It is a small plant popular for studying genetic analysis. A complete genome sequence of A. thaliana is available. It is useful to understand processes including nutrient transport and flower development. CRISPR/Cas 9 gene editing also utilizes this plant.

6.   Mus musculus:

The gene content in mice is similar to that of humans. The genome sequence of mice is known to us. Mouse models are used to study organ and immune systems. Mice are just like humans in developing diseases such as cancer, diabetes, atherosclerosis, hypertension and Alzheimer disease. Thus mouse models are far better to work. The mice help in studying various gene mutations, biochemical pathways, metabolism, pharmacokinetics, and pharmacodynamics. Thus, they are ideal for clinical trials and research.

7.   Neurospora crassa:

It is a haploid fungus widely used in cellular processes. It is possible to grow this strain and use it for tetrad analysis. Typical findings using this model organism involve centromere distance, crossing over and poky mutants. The recessive traits easily show up in the offspring. Thus it is simple to study genetic analysis.

8.   Danio rerio:

The common name for Danio rerio is zebrafish. It is a model organism in stem cell biology and developmental genetics. Its embryos are transparent and help in clearly identifying stages of development. It is easy to feed zebrafish by making a genetic cross.

9.   Lambda bacteriophage:

It is a phage that infects E. coli cells. It replicates using bacterial machinery. A bacteriophage is an important tool for genetic analysis. It is a model system for studying genetic recombination, complementation, and cloning experiments. The lambda genome is capable of insertion into the bacterial chromosome.

10.                      Cavia porcellus:

The common name for this model organism is the guinea pig. However, this organism is not a pig but a rodent. Germ theory was established using guinea pigs as model organisms. They are used to study infectious diseases such as cholera, typhus, and brucellosis.

References:

[1] Biotechnology, David P. Clark, Nanette J. Pazdernik

[2] Introduction to Genetic Analysis, Anthony J.F. Griffiths, Susan R. Wessler, Richard C. Lewontin, Sean B. Carroll

[3] Genetic Analysis: Genes, Genomes, and Networks in Eukaryotes, Philip Mark Meneely

[4] Encyclopedia of Genetics, Eric C.R. Reeve

[5] Neurospora: Contributions of a Model Organism, Rowland H. Davis
[6] Model organisms- Wikipedia

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Expression vectors

Expression vectors express the desired gene within the host. The gene of interest gets inserted into the vector so that it encodes a protein or a peptide. Both the structural genes and promoter sequences get cloned into the same segment of DNA. If a gene gets cloned into a vector, it may or may not express. However, cloning the structural genes with the promoter sequence and then inserting them into the vector helps in the expression of the desired gene. Thus an expression vector consists of a cloned gene, sequences upstream of the cloned genes, and the genes controlling the transcription and translation. The blue-white screening technique helps to check whether the gene gets expressed or not. The expression construct involves a plasmid or a virus designed for gene expressions. Common features of an expression vector involve the origin of replication, a selectable marker, and a suitable insertion site known as multiple cloning sites. It is also known as a polylinker.

Hence, a polylinker is a common site for many restriction enzymes. It gets cleaved by any of the restriction enzymes. The DNA fragment of interest gets inserted in place of the cleaved restriction sites. The gene expression elements present in the expression vector play an important role.

The gene expression elements include a promoter, a translation initiation sequence, a ribosome binding site, a start codon, a stop codon, and translation termination sequences. The expression vector gets designed as per the type of the host. Prokaryotic expression vector consists of sequences suitable for a prokaryotic host. Eukaryotic expression vectors consist of sequences suitable for a eukaryotic host. Shine-Dalgarno sequence belongs to a prokaryotic expression vector. Kozak sequence belongs to a eukaryotic expression vector. The protein purification follows the expression of the genes through an expression vector. Hence, protein purification plays an important role in obtaining the purified product. A purification tag such as a histidine tag, a marker peptide or a fusion partner gets added to a cloned gene. Let us know the examples of the expression vectors in detail.

1. pGEX-3X vector

It is an E. Coli expression vector. The fusion partner involved in this vector is known as glutathione-S-transferase. The gene expression gets controlled through the lac promoter. The pGEX-3X is a 4952 base pair expression vector. It consists of a protein binding site, a lac repressor, a multiple cloning sites, a bla promoter, and a beta-lactamase site. It also consists of the ampicillin resistance gene. The GST enzyme family corresponds to many types of proteins such as the cytosolic, mitochondrial, and microsomal proteins. The expression of the fusion protein gets regulated under the control of the lac promoter. Addition of IPTG induces the protein expression. The GST fusion protein exhibits an ability to bind to a glutathione agarose column. After washing away the unwanted protein, the fusion protein remains as it is. The yield includes 1-10 mg of fusion protein per liter of cells.

 Image: The Expression Vector (pGEX-3X)

2. pMal expression vectors:

It employs a fusion protein known as maltose binding protein (MBP). The lac promoter controls the expression system. The purification of the expression vector involves affinity chromatography on an amylose agarose column. The pMal expression vectors consist of multiple cloning sites and encode oligopeptide proteolytic cleavage site. The vector yields about 5-30 mg of the protein.

3. pET-41a (+) vector:

It consists of a kanamycin resistance gene as a selectable marker. The fusion tag used in this vector is known as glutathione-S-transferase (GST) gene. Unlike other expression vectors, pET-41 a(+) does not have a stop codon after the GST gene. T7 promoter controls the expression of the GST gene and the fusion gene. IPTG (isopropyl -beta-D-thiogalactopyranoside) induces protein expression.

4. Eukaryotic expression vector (pcDNA3):

Introduction of eukaryotic expression plasmid into the cells enables a stable expression. Eukaryotic expression vector requires a specific eukaryotic sequence. A eukaryotic expression vector constitutes three main components such as an ATG codon, a Kozak sequence, and a polyadenylation signal sequence. Transfection process involves electroporation or a calcium chloride treatment.

Applications of expression vectors:

Peptides or proteins get expressed through the expression vector. Recombinant DNA technology helps to derive most of the proteins in the pharmaceutical industry. Hence, they are known as recombinant proteins. Examples include hormones, peptide vaccines, antibiotics, enzymes, antibodies, and protein-based factors. The first human recombinant protein is known as insulin. Screening of cDNA sequences involves expression vectors transformed into bacterial cells grown on a selective medium. Detection of protein-protein interactions involves expression vectors. 
Yeast two-hybrid system is an example of protein-protein interaction. A yeast expression consists of a sequence for Gal4p binding domain fused to a sequence coding a protein. The experiment involves the cotransformation of yeast expression vector with yeast expression library into the yeast cells. Here the fusion protein gets synthesized.

Working model of yeast expression vector:

It involves a yeast galactose metabolism gene GAL1 transcription. The gal4 gene encodes gal4p regulatory protein. The gal4p binds to an upstream activator sequence G. It is an example of a promoter element. Two domains of gal4p include DNA binding domain and an activation domain. The DNA binding domain BD binds to an upstream activator sequence G. An activation domain helps in binding the RNA polymerase to a promoter region. Hence it helps in the initiation of transcription.

Types of expression vectors used in a two-hybrid model:

One type of an expression vector contains gal4p BD sequence fused with a known sequence encoding a protein. The other type of expression vector consists of gal4pAD fused with protein-coding cDNA sequence. Cotransformation of AD and BD plasmids further involve the study of the reporter gene expression.

Production of protein product using an expression vector:

A classic example of recombinant protein production involves a transgenic sheep. An expression vector consisting of a gene of interest must be suitable for the mammalian cells. The beta-lactoglobulin promoter sequence involves the expression of the protein. The protein product isolation becomes simple through milk secretion. The beta-lactoglobulin promoter is an active mammary tissue promoter.

Manipulation of the gene of interest enables its placement adjacent to the beta-lactoglobulin promoter. Hence the expression vectors now get microinjected into the sheep ova. Each ova having the expression vector gets implanted into the foster mother. Identification of transgenic sheep involves PCR analysis. PCR easily detects recombinant sequence. After maturation of the transgenic sheep, the beta-lactoglobulin gets expressed in the mammary tissue. It leads to the production of milk. Separation techniques separate the proteins from the milk.

Other examples of applications of the expression vectors:

·  Human growth hormone production is involved in the pituitary dwarfism treatment

·        Bovine growth hormone increases the cattle and the dairy yields.

References:
[1] Recombinant DNA Technology, Sardul Singh Sandhu

[2] Genetics, 9th Edition (Multicolour Edition), Verma P.S. & Agarwal V.K.

[3] Gene cloning and DNA analysis, T.A. Brown

[4] Genetic Engineering, Verma P.S. & Agarwal V.K.
[5] Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh

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