GENETIC SCREENING: AN ADVANCED AND PROMINENT SYSTEM IN MODERN BIOLOGY
Genetic screening has utilized the important model organism, Danio Rerio, to discover genetic pathways applicable to other vertebrae’s and plant species. Previously unable to study genes, genetic screening has now allowed researchers to link them to species behavior, disease, and development.
Zebra Fish as Model Organisms: Danio Rerio, commonly called Zebrafish, are a relatively new model organism which began to be utilized in the mid-1990s after two large genetic screening projects were completed in Germany and Massachusetts (Lawson, 2011). While Drosophila and C. Elegans prove to be good model organisms, Zebrafish are one of the few vertebrae models that researchers view significant. These are small freshwater tropical fish no larger than 3-4 centimeters that can rapidly develop in 24 hours (Patton, 2001). A large amount of synchronous embryos are produced each year. By one female, nearly 100-200 telolecithal eggs may be produced which are externally fertilized.
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Rapid cleavage occurs in less than 15 minutes (Figure 1). The mid blastula transition takes place around the tenth cleavage. This is when zygotic gene transcription is activated and the parental gene regulation is subsequently turned off. One of the most noteworthy characteristics that makes Zebrafish an important model organism is its’ transparent eggs. This allows gastrulation and organogenesis to be observed under the microscope, along with blood circulation patterns and heartbeat. A young embryos behavior and response to sight and touch is also frequently observed under the microscope. Zebrafish embryos are susceptible to mutagenesis meaning mutations can be induced in high frequency.
Figure One: Above is pictured the early stages of Zebrafish development. (A) Shows the 8 cell cleavage sitting on top of the blastoderm. (B) Blastoderm stage (C) Conclusion of gastrulation (D) Concluding primary axis extension (E) Five somite stage (F) 14 somite stage (G) Embryo at 24 hours (H) Free swimming larva. (Ingham, 2007)
General Genetic Screening Methods:
1. Forward Genetic Screening |
Genetic testing is used to observe specific genes within an organism by overexpression or knockout methods to determine its function. There are two categories in which genetic screening is performed. The most common method is the forward genetic screen in which the zebrafish is consequently mutagenized and then the screen is performed on the organisms that carry the mutation. The second category is reverse genetic screening which will be discussed later. To begin, male parental fish will be treated with a chemical mutagen. Whereas gamma and X-Ray methods were previously used, chemical mutagens are preferred because they can more readily affect specified genes. The chemical typically used is abbreviated ENU in which male fish are submerged. This causes random assortment in germ lines cell and causes mutations (Patton, 2001). A female and the male treated with the chemical mutagen are then mated producing the F1 generation. Fish in the F1 generation may or may not display the mutation depending on whether it is recessive or dominant. The siblings of the F1 generation will be bred to produce the F2 generation. Here, some individuals will carry the mutation while others will not. F2 generations of each family are then mated. Within the F3 generation there is a 25% chance that one of the individuals will carry the mutation (Figure 2). If the mutation affects development it can be observed as this process does not take place within the mother or in a non-transparent shell.
Haploid Screens: While utilizing a chemical mutagen is effective, it is not efficient as it requires thousands of F2 progeny and time is wasted for developing each generation. Haploid screens, therefore, take advantage of the haploid stage of embryos which exists for only a few days. Initially the muta-
Figure 2: Forward genetic screen model for Zebrafish showing the F1, F2, and F3 generations and the possible mutation outcomes. Within the F3 generation 25% are wild-type (+/+), 50% are heterozygous (+/m), and 25% show the recessive mutation (m/m). (Patton, 2001)
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genesis begins the same as previously stated. The male is treated with ENU chemical mutagen by being exposed to water and is then mated with a wild-type female. The F1 generation female is then gently squeezed to speed up the release of her eggs (Patton, 2001). These eggs are fertilized with ultraviolent-treated sperm to produce 50% mutant and 50% heterozygous offspring.
These ratios are much better than the previously stated protocol. UV treatment is able to activate the egg while simultaneously killing the parental DNA (Figure 3ab). By RNA in situ hybridization, brain patterning genes were discovered. Val, for example, is an allele found in Zebrafish responsible for krox20 expression (Patton, 2001). When abnormal expression occurs, an unusually smooth and unsegmented hindbrain results.
Homozygous Diploid Screens: Early pressure (EP) and heat shock (HS) methods are utilized to produce gynogenic embryos. This means that an egg has only maternal chromosomes because it was activated by a sperm that did not enter the cell. Early pressure inhibits Meiosis II from occurring by destroying the meiotic spindles. Mitosis begins with a diploid cell. In heat shock treatment Meiosis I and II are performed as usual. The egg is exposed to UV-treated sperm, causing the haploid cell to skip its first mitotic division and enter directly into the second (Figure 3c). This type of screening has become essential in identifying genes involved with motor neurons. Deadly Seven (des) motor neuron mutation causes irregular somite formation. Des is responsible for programming anterior-posterior and myotome formation of somite’s in synch the development of the nervous system (Patton, 2001).
Figure 3: The diagram shows the systems for haploid and homozygous diploid screens. In (A) a wild-type female and ENU treated male are mated. F1 is exposed to UV treated sperm which does not enter the nucleus. In only the F2 generation, mutant species can be observed. (B) shows phenotypic differences between a normal zebrafish and one that was treated with the UV sperm. In (C) the processes for early pressure and heat shock is shown. (Patton, 2001)
Phenotyping: While many forward screens use observation to discover the effect of the gene on the embryo, assays and more detailed mutant screening analysis has been developed.
Fluorescence and Molecular Markers:
One of these ways is through molecular markers on specific cells or tissues. Rag1, for example, is a molecular probe that has been utilized to determine genes that effect thymus and lymphoid cell development when mutated (Lawson, 2011). Phospholipid probes have also been used when visual screening proves to be inaccurate. These probes aid in determining mutations in digestive tract functioning. When fed to a larva of the F3 generation, through fluorescence, a fat free mutation was able to be identified, which was not an obvious physical defect in the gut. After cloning the gene, it was concluded that it was essential for Golgi trafficking and lipid absorption (Lawson, 2011). In addition to these molecular markers, GFP (Green Fluorescent Protein) has been utilized in screening.
Behavior Screening: Post mutagenesis, behavior screening observes the organism most commonly based on its response to touch or visual stimulation. Using a behavioral assay, the space cadet (spc) mutation was revealed when fish did not perform their usual “fast-fleeing” response to stimuli. From this observation, it is concluded that the spc gene is responsible for the axon trajectories that controls locomotion (Patton, 2001). In another interesting discovery, researchers have been able to breed mutants that are not susceptible to cocaine addiction. Goody-two-shoes (gts), dumbfish (dum), and jumpy (jpy) may provide understanding in the brains dopaminergic signaling pathways (Patton, 2001).
2. Reverse Genetic Screening The process eliminates or reduces the expression of a gene instead of searching for one as in forward genetic screening. Healthy males are made into mutants using the chemical mutagen ENU and then are mated with wild-type females. The DNA can then be examined for mutations by TILLING or sequencing.
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Targeting Induced Local Lesions in Genomes (TILLING): Sperm from F1 males is cryopreserved and DNA is secluded and saved for lesion analysis. Lesions are identified through CEL 1 assay which is an endonuclease cleaving the DNA and then sequencing the region. The preserved sperm is then used to create the F2 generation in which half will carry the specified gene. TILLING essentially produces mutations in a family of organisms in many genes (Figure 4). Rag1, for example, has been studied through the reverse genetic screening system of TILLING. Typically this gene aids in the functioning and development of T and B cells. Mutant fish are surprisingly not changed by alteration of Rag1 leading to the conclusion that they may become vital in transplants (Lawson, 2011). Disease related alleles have been found using this process notably in mutants vhl and tp53 used to test cancer biology (Lawson, 2011).
Screening Applied to Human Disease:
Genetic screening has allowed researchers to link many genes to human disorders. Through biochemical screening, the genes responsible for coagulation have been discovered and tested. Coagulation is very important during injury in humans, and coagulation disorders may include hemophilia, thrombosis, and hemorrhage. In mammals, to cleave fibrinogen, factor VII must be activated to create a cascade of proteases. By cutting the tails of zebrafish and testing their blood by turbidometry, eight mutant genes were discovered (Patton, 2001). Reconstitution assays are still currently
Figure 4: This displays the process of TILLING in reverse genetic screening. F1 generation is created from a wild-type female and ENU treated male. Under PCR amplification, exons are intensified and then screened by CEL 1. (Lawson, 2011)
being performed in hopes of identifying and understanding the similarities between this model organism and humans.
Conclusion: Forward genetic screening and reverse genetic screening both provide a unique and beneficial understanding of genes linking to mutations. Genetic screens in zebrafish provide great information into development and disease which can then be applied to humans. Genetic screening is a technique that is still being perfected today.
Ingham, P. “Zebrafish Genetics and Its Implications for Understanding Vertebrate Development”. Oxford Journals. 10. (2007): 1755-1760. Web.
Lawson, N. and Wolfe, S. “Forward and Reverse Genetic Approaches for the Analysis of Vertebrate Development in the Zebrafish”. Cell Press. 21. (2011): 48-64. Web.
Patton, E. and Zon, L. “The Art and Design of Genetic Screens: Zebrafish”. MacMillian Magazine. 2. (2001): 956-966. Web.