Zebrafish as a model: Introduction of genetics to undergraduates in a large laboratory class

Abstract

Laboratory activities based on zebrafish, Danio rerio and nematodes have been created for a more comprehensive integrated biological laboratory course. The course will is meant to enable students examine zebrafish at its various developmental phases and take note of  shifts taking place  in response  to   the interested genes, that is the Pax6a and Pax2a within an adult zebra fish brain. A six week laboratory module was made based on examinations methods including bioinformatics. Students are expected to perform various sets of experiments including observation of a zebrafish in its natural habitat and at its stages of development or embryogenesis and important dye staining of mutants. Activities and procedures carried out in the laboratories are supposed to equip the students with hands-on skills, increase research oriented information and enhance their reporting writing skills and oral presentations (Detrich, Westerfield &Zon, 2011).

This intensive six week course for undergraduate students will aim at development and genetic makeup of zebrafish. It will cover standard and novel technology focused toward their application in zebrafish .Morning and evening will be devoted to laboratory procedures and evening will be devoted to discussions and lectures Detrich, Westerfield &Zon, 2011).

The lectures and laboratory work will introduce students to development of the zebrafish and methodologies for manipulation and examining gene functionality   such as genetic and micro molecule screening, mapping and cloning; mRNA approaches and cloning (Detrich, Westerfield &Zon, 2011).

 

 

 

The main objectives of this course

The wide field of development biology and genetics is highly interdisciplinary with all the fundamental scientific sections as wells plant and organism biology. Its main objective lies in assisting students acquire a systematic understanding of the processes cells undergo to achieve their various fates and the combination of intercellular signals and intracellular regulating circuits encoded within the body. The course will be engaging the students in realistic research while giving them essential skills and concepts required to achieve a  their student objectives (Determination of Expression of pax6a and  Pax2a in adult Zebrafish Brain.PDF

 

 

 

Learning objectives of genetic lab

To demonstrate knowledge and understanding of genes Pax6a and Pax2a within the zebrafish development stages. To be evaluate, accurately interpret and participate in reporting the results from the experiments conducted in the laboratory. To assist in developing knowledge and understanding in dealing with advanced specialized biological topics. Zebrafish are vertebrates hence they have a high degree of similarities and functions with mammals including humans.

 

Introduction

             Standard set of experiments will be carried out to help researchers study the expression of a gene that exists within a specific tissue type. The first laboratory tests will   focus on working on the RNA and it separation   through the organic extraction methodology .Trizol will be used to conduct   this experiment Trizon. Function as a reagent  that  isolates a protein from its nucleic acid .Subsequently, the extraction of  organic biological materials provides a chance  for further separation  of the RNA form the DNA .After the RNA is separated from the DNA ,reverse transcriptase-polymerase chain reactions is  conducted. Reverse transcriptase polymerase chain reactions are variants of the PCR that can be conducted founder the Murder Mystery Laboratories. This type of variant of a PCR is normally carried out in a Murder Mystery Laboratories. In this type of variant, enzyme reverse transcriptase to create a corresponding DNA version of the RNA. CDNA is used in the polymerase chains reactions to upgrade particular genes of interests .Agarose gel determines whether amplifying of particular genes of interest was successful or failed. The PCR product is placed within circular pieces of the DNA   known as the Plasmid (Determination of Expression of pax6a and Pax2a in adult Zebrafish Brain.PDF).

 

 

Genes of Interest

Pa6a and pax2a are responsible for the differentiating of cells, the migrating of neural cells and the maintenance of the neural cells. They are categorized as transcription factors .Transcriptors bring together other proteins that will ensure other functionality of other genres are turned off hence they cannot express themselves. The Pa6a and Pax2a   are made up of DNA binding domain known as the paired domain (Determination of Expression of pax6a and Pax2a in adult Zebrafish Brain.PDF).

Pax6a or the master regulator   forms the eye. When Pax6a homologs are expressed deliberately i.e. inserted in an   organism’s cells, eyes will be generated instantly.Pax6a is an indicator for stem neural cell. It develops neurons located at the forebrain   and midbrain of each and very organism. Any mutations within the brain or eyes are as a result of lack of pax6a regulator (Determination of Expression of pax6a and Pax2a in adult Zebrafish Brain.PDF).

Pax6a develops vital organs such as the optic nerve, ear and the kidney and the cerebellum. However when introduced within the human body, it is not enough to form these vital organs. In a human heterozygous mutation leads to hole in the eye: coloboma, hearing loss and kidney failure (Determination of Expression of pax6a and Pax2a in adult Zebrafish Brain.PDF).

The expressions of   Pax6a and Pa2a are antagonizing to each other’s expression .Hence they cannot be expressed in the same cell. Whereas pax2a is expressed in the optic nerve where they transmit visual information, the Pax6a is expressed in the retina, where it converts light into pictures. When Pa2a is absent, retina retreats into the optic nerve region and when Pax6a is absent, it retreats to the eye forming a blind spot in the middle part of the eye (Determination of Expression of pax6a and Pax2a in adult Zebrafish Brain.PDF).

RNA Extraction methodology

Liquid-liquid extracting method is used for the separation of mixtures of various molecules. The different solubilities of each molecule in two v variant immiscible liquids. Liquid-liquid extraction is used to separate RNA, DNA and proteins. Kirby realized that nucleic acids can be isolated from proteins in 1956. The same procedure can be used to isolate RNA (Determination of Expression of pax6a and Pax2a in adult Zebrafish Brain.PDF).

 

RNA extraction methodology

Use a syringe to homogenize the tissues then pipetting up and down.1 brain per 750ul of the trizol .The syringe is placed in a sharp container the reaction is incubated at room temperature for 5minutes.02ml chloroform is added in an inverted c tube. The tube is then closed for 30 seconds and then incubated at room temperature for 15minutes.centrifuging is done at a speed of 12k for a duration of 15mintues. The aqueous is then extracted.0.5ml isopropyl alcohol is included in the colorless ad mixed. Incubation is repeated for ten minutes at room temperature. Ice is placed on the resultant solution for ten minutes. Pining is done yet again at speed of 12k for ten minutes .RNA are expected to appear as white pellets at this point. Supernatant are removed and washed with 0.5ml of cold 75 ethanol (Determination of Expression of pax6a and Pax2a in adult Zebrafish Brain.PDF).

 The pellets are then dried by invert on of the tube on paper towel for a duration of ten minutes. The dried pellets are then dissolved in 150ul pure water and put in water bath at a temperature of 55c for around ten minutes.20ul of 10m NH4OAC is added to the resulting solution. Then removed with chloroform .After the aqueous phase is subtracted 100% of ethanol is added to the mixture and labelled. Incubation is done at -80 for a duration of a week (Determination of Expression of pax6a and Pax2a in adult Zebrafish Brain.PDF).

 

 

RNA extraction and RT-PCR

Spinning at 12k for around 15minutes then remove the supernatant .The pellets should be washed with 500ml of 75% of cold ethanol. The results should be spanned at a speed of 12k for a duration of 5minutes.The supernatant is then removed and dried for 10minutes in a towel paper (Determination of Expression of pax6a and Pax2a in adult Zebrafish Brain.PDF).

cDNA synthesis

5 ul RNA template is put in a centrifuge tube, 1 ul oligo primer and 1 ul if dNTP.it is stored at a temperature of 65c for 5minutes.incubation is done in ice at temperature for 1 minute. The results are then centrifuged in a tube. The solution is then pipetted up and down and spinned shortly in a centrifuge. Incubation is done for at a temperature of 50c for 30 minutes. Incubation is repeated for 15minutes at temperature of 70c (Determination of Expression of pax6a and Pax2a in adult Zebrafish Brain.PDF).

Polymerase chain reaction.

These are the reagents needed when using the bioline protocol:

2.5ul 10x PCR buffer

2ul MgCl2

0.5ul forward primer and reverse primer

0.5 taq polymerase

5ul cDNA created in previous

1ul NTP

13 water

The PCR is run through the thermocycler by utilizing program at 95c for around 5minutes and 30sec, 95c-30sec and 55c-60sec (Determination of Expression of pax6a and Pax2a in adult Zebrafish Brain.PDF).

 

 

Worksheet for lab 7 and 8

Question 1

Use detergent and wash the hands thoroughly.

Question 2

 They should be completed in a fume chamber

Question3

It settles at the bottom of the immiscible mixture.

Question 4

Transcriptase

Question 5

cDNA

Question 6

Trizon.

Question 7

Plasmid.

Question 8

 It indicates that there is DNA replication with in the cells (Determination of Expression of pax6a and Pax2a in adult Zebrafish Brain.PDF).

 

Bacterial cloning

This is TA technique used for sub cloning that limits usage of restriction types of enzymes. It is quicker than other methods of sub cloning .It entirely relies on the ability of adenine and thymine which are complimentary pairs within the Various DNA pieces. This happens in the presence of ligase (Bioinformatics Exercise 2.PDF).

Methodology of the isolation of DNA

This procedure makes use of bacteria for the production of large amounts of plasmid. The plasmid is then extracted for evaluation. This process makes use of   column extraction kits known as mini prep kits. They are available commercially. The process is centered on centrifugation which separates plasmid from the rest of bacterial genome. During   carrying out the procedure gloves are worn to avoid the irritant chemicals (Bioinformatics Exercise 2.PDF).

 

 

 

Methodology of restricting digest with Ecorl for verification inserting sizes

Double stranded DNAs are cut at particular regions by restriction enzymes. The enzymes are protein in nature. Scientists W.Arber and D.Dussoix were the first individuals to use the enzymes in the 1960. They used it to explain bacteriophages i.e. bacteria that are infected by viruses but do not affect others. Restriction enzymes are normally separated from bacteria (Bioinformatics Exercise 2.PDF).

Methodology of Agarose Gel Electrophresis.

It is done under three stages: Microwaved until it melts and more clear or colorless and then cooled. For 3 minutes. Gel is then poured into a cassette. The gel is then run for 30 minute duration .It is then imaged (Bioinformatics Exercise 2.PDF).

Sequencing

In Sanger sequencing, a primer is utilized to begin replicating from one strand of the DNA to other. In our context, the plasmid contains two sequences t flanking the inserts. They are known as T7 and SP6.In addition to the conventional nucleotides, when they are integrated, they play an important role of preventing   any further amplification of any specific strands. Dye terminator nucleotide is usually added to the normal nucleotides. DNA strands of different lengths can be isolated through the process of electrophoresis. This is conducted when various fluorophores are placed on different bases .The reaction can be interpreted by a capillary gel i.e. contained in one lane. The smallest among the fragments move faster within the gel whereas linger strands take more time to get through to the detector. This produces to the output (Bioinformatics Exercise 2.PDF).

Analyzing the sequences.

First open the first sequence file within the APE. It opens the chromatogram. The colored lines should correspond to each and very dye as they are passed through the detector. Intensity is illustrated on the Y access. The blue shadings usually represent quality of the sequencing reads or interpretations. The initial readings are usually of a lower quality and it improves as it is progressing into the sequences. This a general phenomenon due to lack of better technology (Bioinformatics Exercise 2.PDF).

Primer Design

Separated fragments are just but a small portion of a gene. Small fragments of PCR products are good for deciding on expressions. To get more Valuable results can be improved when pax6 and pax2 (Bioinformatics Exercise 2.PDF).

Bioinformatics exercise 2

This is the use of computer software and information technology to research on biological systems and other scientific phenomena. This a very vast field or profession and includes various  topics such as drug designs and protein folding .Many computer programs for instance, Java and SQL  can be used for biological study purposes and other  aspects  of the field (Bioinformatics Exercise 2.PDF)..

Being familiarized with bioinformatics tools is very useful in the biological field as it helps understand vital modernized concepts such as molecular biology and DNA cloning and replication of the RNA. Even other files of biology such as ecologists can make use of bioinformatics and other specified fields of biology which are dependent on computer programming and information technology (Bioinformatics Exercise 2.PDF).

PubMed is a database that has all biological information in terms of literature. This Literature is hosted at the Ana institution known as Biotechnology Information. Since DNA sequencing was discovered, the amount of data that came with was enormous and exploded hence the need for database. All known DNA sequences are arranged in a catalogue at NCBI.The data base that has all of the genetic sequences that are used to study other components yet discovered .Gen Bank is the database. By the year 2008, Gen Bank had already listed more than 100billion base pairs that is, their nucleotides and their corresponding partners. The rate at which the data increases per year is very fast than the theory of Moore’s Law i.e. the ideology that the numbers of transistors non a computer’s chip, complimentary to speed would increase twice the number of the previous year due to the growing and advancing DNA sequencing which has contributed largely to this fact (Bioinformatics Exercise 2.PDF).

Scientists use a lot of tools to look up information. Using the link http://www.ncbi.nlm.nih.gov, against drop down list one can choose ‘all databases’. An icon is next to search bar. Then type Tubulin and process the search button. This page will give the amount of times one has searched for an item.it will also reveal the number of articles that are related to the tubulin the researched word .Therefore it is very systematic and orderly (Bioinformatics Exercise 2.PDF).

Each of the databases have highly valuable information and can help one to advance in their careers. It is helpful to learn the types of information that are contained in the databases. The link   at the top of the website can be used to show older book editions. It is labeled ‘books’. It can be used for referencing of the books. For people who are interested in the field of medicine of needs to use the GTR link. The link leads to a gene tests registries. Clicking on the GTR label it opens up the beta tubulin which consequentially leads to a list of hospitals laboratories that provide gene tests for specific disorders and can be easily compared (Bioinformatics Exercise 2.PDF).

Going back to the home database page. Conserved proteins domain are sections of a protein that can be folded independently and are normally associated with a certain role. On the conserved domain one can look up the definition portrait above. Present in the tubulin are the conserved domains (Bioinformatics Exercise 2.PDF).

There is the gene page. Click on a link labelled gene. The page contains curate of other sections of the database and arranges them into a more comprehensive summary of gene topics. When one clicks on gene, the results can be picked from 29000 genes. This can be singled out by typing TUBA1A.Now one can view only pick one alpha tubulin gene. When one looks keenly look at the list, it is discovered that there are various alpha tubulin. For example: Homo sapiens. Mus muscles etc (Bioinformatics Exercise 2.PDF).

 This is the table that tabulates the developmental stages of a zebrafish laboratory modules which is arranged in terms of weeks corresponding to the specific activities that will be carried out.

Week1	Lesson: Fundamentals  to Zebra fish
Week 2	Observing a live zebra fish and how it develops from conception
Week 3	Mutation and their developments:1defining a mutant by analyzing its arrangement of gene expressions in the wild kinds and deviation
Week 4	Mutations and developments two: blotting using critical dyes to define morphological differences within wild categories and mutant.
Week 5	Environmental impacts on development
Week 6	Laboratories group presentation

 

Conclusion

Introduction to biology laboratory course work takes a step by step approach in the field of experimental biology and genetics.This course has utilized the zebra fish exemplary system that fitted in well with this kind of laboratory course work. The introductory course does a good job and address all other issues that previous similar courses failed to do. It is relevant and interesting and gives a new perspective to genetics due to its engaging experiments. In addition, this course can be termed helpful and can be applied to other laboratory courses.

 

 

 

 

 

 

 

 

 

 

 

 

References

Bioinformatics Exercise 2.PDF

Determination of Expression of pax6a and

Pax2a in adult Zebrafish Brain.PDF

Detrich, H. W., Westerfield, M., & Zon, L. I. (2011). The zebrafish: Genetics, genomics and informatics. Amsterdam [Netherlands: Elsevier/Academic.