Schoen,+Tyler

Zebrafish genetics


 * Lab 1 Isolation of zebrafish genomic DNA**

1. First the zebrafish was completely sedated with 200mg/L tricaine, 2. The fish was sacraficed via scalpel decapitation. I sectioned the fish into many smaller diced pieces and then with liquid nitrogen the contents of the fish were snap frozen. 3. After freezing the fish pieces, we immediately began to crush the contents with mortar and pestle. When a fine powder of fish contents was finally we added the remains to 7.5 mL of lysis buffer in a 50 mL conical tube. 4. We then allowed the solution to incubate for 45 min on a rocking room temperature platform. 5. Once finished 18mL of ethanol was slowly added to the solution to separate the DNA from the two layers. 6. The DNA was then extracted using a self made fire burned glass hook and placed within a microfuge tube with 1mL of TE. 7. The DNA was then stored in the refrigerator for further examination and use in the Genomic DNA characterization in the next lab period.

Lab 1 Identification of conserved non-coding (putative enhancer) sequences

GATA4 gene CNS Location- chr20:60,780,457-60,780,541

CAGCATTAACATATCCTGCAATAGTATGATCCAACATTAGGATAATCCAG CATTAATATATATTTAACATTAGCATATCTAACATTAGCATTCCTGAAAT TAGCAAATCCCAAAATTTGCATATCCTTACAGTAGCATATCCCACTGCAC CTGTCTATCTCACCTGGCTGTATCTGGCATTC[ TCCTTCAGGTGGACGCCG ATGCATCTGGTGATCTGCAGAAGCCAACTCCACTGTGTCTGCAGCGTCTC CATGTATGCCTGCCCA ]AAACACAGACAAAAAAAAGAATTGATTAATTCAT GAGTCATGATTCTTTCCTGAGGGATTCTGAATCGATTCCTAAAAGCCTAC ATATACTGCAGAGTTTGCTTTATTTGGACAAAAAAAGGACTATCTTTAGA ATTTTAAAGATGTGGCAAAAGTGAAATGTTGATTTGTACGTCACTTTCGA CTTGTTTTCTGACT

Vista Browser- Visual of Sequence compared with human genome 1. Using the Vista Browser I compared the sequenced genome of a human with a zebrafish and specifically analyzed the gata4 gene. The comparison is displayed below. 2. Once the proper area was designated that displayed the best correspondence between the two genomes that section was selected for and two primers on each side of the conserved noncoding sequence were designed using the Primer3 output software.

Primer Design

Left Primer- ACCTGGCTGTATCTGGCATT Temp 59.58 degrees Celsius Start 162 Length 20 Right Primer- TCAGAATCCCTCAGGAAAGAA Temp 58.85 degrees Celsius Start 330 Length 21 sequence size 464 PRODUCT SIZE: 169

In Silico PCR

Primer3 Output of genomic sequence and primer




 * Lab 2 Genomic DNA characterization and PCR**

1. I began by preparing a .5% agarose gel by heating 25g agarose in 50mL 1xTAE buffer in the microwave until the powder was completely dissolved. Then a .5x concentration of of GelGreen DNA stain was added to the agarose and stirred into the mixture. The gel was poured out to create the mold and the comb was stuck into the gel to create the boxes for DNA samples.

2. Once the gel hardened the comb was removed and the gel was covered with 1x TAE buffer within the electrophoresis rig.


 * Lab 3 Proximal promoter PCR amplification & agarose gel extraction**

Our oligonucleotide primers were designed and order from Integrated DNA Technologies, INC. The primers were freezdried for stability at room temperature.I briefly spun the microfuge tube containing your oligonucleotide primer to ensure the material was at the bottom of the tube. Then diluted the primers in biology grade water to create a 100 micromoler stock solution of each primer. The molarity was calculated by multiplying the nanomols by 10. The found value was the appropriate microliter amount that was added to each tube. These were then vortexed again to bring the solution down in the tube. Created a 10 micromolar primer working solution of each primer by adding 10 micro liters of the forward and reverse primer to 80 micro liters of biology grade water and briefly vortex. The working solution was then stored within the freezer at -20 degrees celcius.

The PCR was run using the Bio-Rad thermocycler with the annealing temperatures set at 59, 58, 56.5, and 54°C.

(to characterize the reaction specificity and confirm the amplicon length is correct) 1. Made a 2% agarose gel with 50ml of 1xTAE solution and 1g or agarose gel concentration 2. heated until all agarose concentrate was dissolved 3. added 2.5µL of GelGreen to solution 4. poured gel and let stand for 10 mins 5. covered gel apparatus with 1xTAE solution 6. added 5µl of 100bp generuler to second loading lane 7. mixed 5ul my zebra fish DNA with 1ul of tritrack 6x loading dye 8. ran the gel for approximately one hour at 110v
 * Gel Electrophoresis**

1. added 3 to 1 ration of QX1 buffer to the gel 2.allowed to incubate at 55 C for 5 minutes 3.vortexed the QIAEX II suspension for 30 seconds 4. added 10ul of QIAEX II to gel solution 5. incubated at 50 C for 10mins to allow the agarose to solubilize and DNA to bind to silica beads 6. vortexed every 2 mins 7. centrifuged for 30 seconds and discarded supernatant 8. washed pellets with 500ul QX1 wash buffer, vortexed, centrifuged and removed supernatant which removed residual agarose 9.washed pellet twice with 500ul of wash buffer PE, resuspended by vortexing, pelleted with brief centrifugation, and removed supernatant to remove the excess salt 10. air dried the pellet for 10 minutes 11. eluded DNA by resuspended in 30ul of TE buffer and incubated for 55 C for 5 minutes 12. spun the tube and removed supernatant which now contains the DNA 13.stored at -20 C
 * Gel Extraction**

1. added 1ul of BP clonase reaction to microfuge tube and placed on ice 2. Thawed TOP 10 cells and added 20ul to the BP clonase reaction 3. incubated on ice for 30mins 4. heat shock DNA into the cells for 30s at 42 C 5. put heat shocked sells back on ice for 2mins 6. aseptically over flame added 250ul of S.O.C medium to cells 7. shook cells for 37 C for an hour 8. spread 200ul of transformed cells onto prewarmed kanamycin plates 9. incubated plates overnight
 * Lab 4 BP clonase reaction**

//Despite years of intense investigation, cardiovascular disease remains the leading// //cause of death in the industrialized world. The development of new therapeutic// //strategies is hindered by an impoverished understanding of the molecular mechanisms// //that control cardiovascular development and remodeling. The overarching// //goal of this investigation is to elucidate key molecular mechanisms of heart development// //and pathophysiology.// //The visualization of temporal and spatial gene expression patterns is foundational// //for understanding the role of genes during development. While indirect immunofluorescence// //and in situ hybridization techniques provide atemporal snapshots of gene// //expression at the protein and RNA level respectively, spatial and temporal examination// //of gene expression patterns during development is limited to the availability of// //antibodies or nucleic acid probes. Using transgenic fluorescent reporter genes such// as green fluorescent protein (from jellyfish) or the mCherry protein (a red fluorescent protein derived from //Discosoma// coral) gene expression patterns may be monitored in living embryos using non-invasive techniques. Alternatively, the β-galactosidase reporter (a bacterial enzyme that catalyses a colorless substrate into a blue pigment) allows for increased sensitivity over the conventional techniques listed above. Furthermore, conventional techniques do not allow one to isolate specific gene enhancers or promoters during development. The specific aim of this study is to characterize the regulatory enhancer sequences of genes (GATA4, MEF2C, SRF, and TBX5) known to regulate the growth and differentiation of heart muscle cells. While the importance of these genes in heart development is already appreciated, the specific timing and anatomical contribution of these genes to various structures of the heart is poorly understood. This study should shed light on these fundamental mechanisms. Regulatory sequences that may control the expression of these genes will be identified using a bioinformatics (computer-based) approach, amplified from zebrafish genomic DNA (using polymerase chain reaction), and recombined with transgenic fluorescent reporter genes (using recombinant DNA technology). These constructs will then be injected into zebrafish eggs to visualize the gene expression patterns during development of the fish. Traditional methods for cloning gene regulatory promoter and enhancer sequences for analysis are time-consuming and limited by available restriction sites. The //att// site-specific recombination system from bacteriophage λ provides a high-efficiency and high-fidelity genetic cloning system which may be used to bypass these limitations. 1 The bacteriophage λ integrase enzymes used to mediate this reaction are now commercially available from Invitrogen as the “Gateway Cloning System.”2 Historically, the efficiency of zebrafish transgenesis was limited by the low frequency of genomic integration in microinjected zebrafish embryos. This limitation may also be bypassed by the recent discovery of the Tol2 transposon-based system.3 Subsequently, the laboratories of Chi-Bin Chien and Nathan Lawson have made available various clones containing both green and red fluorescent reporter proteins, useful for the characterization of gene-regulatory sequences.4,5
 * //1. Overview of transgenesis strategy//**


 * //Characterization of genomic DNA quality by agarose gel electrophoresis//**

DNA yield can be measured by either spectrophotometric absorbance at 260nm or agarose gel electrophoresis relative to known standards. Estimation of genomic DNA concentration by electrophoresis is generally considered superior as absorb- ance readings may be artificially high due to the presence of contaminating UV- absorbing material. Additionally, electrophoretic characterization allows for estima- tion of the average length of genomic DNA relative to known standards


 * //Introduction to polymerase chain reaction//**
 * //(PCR)//**

In 1983, Kary Mullis at Cetus Corporation developed a molecular biology technique that has since revolutionized genetic research and earned him the Nobel Prize in 1993. This technique, termed the polymer- ase chain reaction (PCR), was rapidly adopted as a significant multidisciplinary research tool. Before the invention of PCR, techniques for genetic analysis were labor intensive, time consuming, and required a high level of technical expertise. PCR has contributed to the development and popularization of gene maping, gene cloning, DNA sequencing, and gene detection technology.

The objective of PCR is to produce a relatively large amount of a specific piece of DNA from a small amount of nonspecific DNA. Technically speaking, this means the controlled enzy- matic amplification of a template DNA molecule containing a specific DNA se- quence of interest. A researcher PCR to amplify trace amounts of DNA from a drop of blood or a single hair follicle to generate millions of copies of a desired DNA fragment. In theory, only one template strand is needed to generate millions of new DNA molecules. Prior to PCR, genetic and forensic analysis required copious amounts of DNA.

PCR Makes Use of Two Basic Processes in Molecular Genetics: 1. Complementary DNA strand hybridization 2. DNA strand synthesis via DNA polymerase

Before a region of DNA can be amplified, one must identify and determine the se- quence of a piece of DNA upstream and downstream of the region of interest. The- se areas are then used to make the **oligonucleotide primers** that will serve as starting points for DNA replication. Again, primers are needed because **DNA poly-** tiate replication of DNA or synthesize new copies of template DNA. The two strands of the fragment of interest will be melted apart into single strands before synthesis begins. Therefore, primers are required to provide a double-stranded start point for the DNA polymerase.
 * merases** require double-stranded DNA (as opposed to single stranded DNA) to ini-

The DNA polymerase used in PCR, however, must be a thermally stable polymerase because the polymerase chain reaction cycles between temperatures of ~60°C and ~94°C. A thermostable DNA polymerase (//Taq// polymerase) from the thermophilic bacterium, //Thermus aquaticus//, is commonly used for this purpose. Alternatively, it has been recently demonstrated that the DNA polymerase (//Pyro// polymerase) from the archaebacterium //Pyrolobus fumarius// while equally thermostable, retains activity longer and demonstrates greater processivity (average number of nucleotides add- ed per association).

Following sample preparation, the template DNA, oligonucleotide primers, thermo- stable DNA polymerase, the four deoxynucleotides (A, T, G, C), and reaction buffer are mixed in a single microfuge tube. The tube is placed into the thermal cycler. Thermal cyclers contain an aluminum block that holds the samples and can be rap- idly heated and cooled across extreme temperature differences. The first step of the PCR temperature cycling procedure involves heating the sample to 94°C. At this high temperature the template strands separate (denature). This is called the **dena-** allow the primers to anneal to the separated template strands. This is called the **an-** reanneal to each other or compete with the primers for the primers complementary binding site. However, the oligonucleotide primers are added in excess such that the primers actually out compete the original DNA strand for the primers' comple- mentary binding sites. Lastly, the thermal cycler heats the sample to 72°C for the DNA polymerase to extend the primers and make complete copies of each template DNA strand. Becaues the polymerase works most efficiently at this temperature it is called the **extension step**. Two new copies of each complementary strand are cre- ated. There are now two sets of template strands. These two sets of template strands can now be used for another temperature/thermal cycle and subsequent strand synthesis. At this stage, a complete temperature cycle (thermal cycle) has been completed (Figure 2). Thermal cycling continues for 40 cycles. After each thermal cycle the number of template strands doubles, resulting in an exponential increase in the number of template DNA strands. After 40 cycles there will be 1.1 x 1012 more copies of the original number of template DNA molecules. The most unique feature of PCR is the generation of a precise length and sequence of DNA. On the first cycle the two dif- ferent oligonucleotide primers anneal to the original genomic template DNA strands at opposite ends and on opposite strands. After the first complete temperature cy- cle, two new strands are generated that are shorter than the original template strands but still longer than the length of the DNA that the researcher wants to am- plify. It isn’t until the third thermal cycle that fragments of the precise length are generated (see Figure 3).
 * turation step**. The thermal cycler then rapidly drops the temperature to 50-60°C to
 * nealing step**. There is the possibility that the two original template strands will
 * Temperature cycle = denaturation step + annealing step + extension step**

It is the template strands of the precise length that are amplified exponentially (Xn, where X = the number of original template strands and n = the number of cycles). There is always one set of original long-template DNA molecules which is never fully duplicated. After each thermal cycle, two intermediate length strands are produced, but because they can only be generated from the original template strands, the in- termediate strands are not exponentially amplified. It is the precise length strands generated from the intermediate strands that amplify exponentially at each cycle. Therefore, if 20 thermal cycles were conducted from one double stranded DNA molecule, there would be 1 set of original genomic template DNA strands, 20 sets of intermediate template strands, and 1,048,555 sets of precise length template strands. After 40 cycles there would be 1 set of original genomic template DNA strands, 40 sets of intermediate template strands, and 1.1 x 1012 sets of precise length template strands (see Figure 4).

//Morpholinos are ~25bp oligonucleotides that are designed to hybridize to the translation// //initiation site (5’UTR) of target mRNAs to sterically block ribosome-mediated// //translation. The backbone of morpholinos is chemically modified to be non-ionic.// //This is thought to minimize potential interactions with proteins. The backbone of the// //morpholino is further modified by replacing the suger (ribose or deoxyribose) with a// //morpholino ring (see Figure 1).//
 * //Introduction to gene knockdown using morpholinos//**

LAB REPORT

GATA4, a gene that encodes a zinc-finger transcription factor crucial to embryogenesis and myocardial differentiation and function has been associated with cardiac septal defects in the atrium. Further knowledge of the Gata4 gene could lead to understanding aspects of human heart development and atria septal malformation. Unfortunately unsuccessful in my efforts, I intended to design and construct a transgenic zebra fish via sequenced steps including design and amplification of a putative promoter, gateway clonase in bacterium and embryonic injection of the transgenic gene. The results of these techniques display a transgenic zebra fish that expresses GFP when driven by a GATA4 putative promoter. This will allow a greater comprehension of GATA4 expression in the developing zebrafish, which directly correlates to the genes expression in human heart development.


 * __ Introduction __**

Research in the area of human heart development has been associated with and includes work to further understand the expression of the GATA4 gene. GATA4 encodes two zinc transcription factors that bind to a consensus sequence in the production of proteins associated with cardiac myocyte enlargement. The mutation of GATA4 has been directly correlated with patients exhibiting congenital atrial septal defects. (Wang et al. 2010). GATA4 defects have been determined to cause atrial septal defect type 2, which is the congenital heart malformation characterized by incomplete closure of the wall between the atria resulting in blood flow from the left to the right atria. Patients with atrial septal defects also display other heart abnormalities, which include ventricular and atrioventricular septal flaws, pulmonary valve thickening, or insufficient function of cardiac valves (Garg et al 2003). Research of GATA4 using transgenic zebrafish provides the optimum technique in visualization and characterization of the expression patterns of GATA4 throughout zebra fish growth and development. Obtaining this information can create a better understanding of GATA4s role in heart development. Because GATA4 is homologue to the human GATA4 gene, information from the zebrafish expression pattern can be associated with the genes expression in humans as well, making the research valuable. A sequence of detailed laboratory techniques was performed to discover a better understanding of the GATA4 gene. The major steps in the process of creating the transgenic fish include: genomic DNA isolation, primer design, amplification of the putative promoter using polymerase chain reaction, gel electrophoresis extraction, gateway vector cloning using BP clonase, sub cloning into an expression vector using LR clonase, and microinjection into zebrafish embryos. The goal of the research is to produce and monitor a transgenic zebrafish that displays a green fluorescence from the GFP that has been encoded into the zebrafishes genome. The fluorescence will show where and when the GATA4 gene is being expressed in the heart and possibly in other tissues or organs. Recent findings demonstrate that GATA4 mutation has been associated with decreased transcriptional activity and provide insight into a pathogenic link between GATA4 function and congenital atrial septal defects (Liu et al. 2011). In producing a transgenic zebrafish I hope to further our understanding of the molecular mechanisms involved in GATA4 expression and its relation to human heart development.


 * __ Methods __**

Cardiovascular disease remains the leading cause of death in the industrialized world. The development of new medical strategies has been fighting the many obstacles in understanding the molecular mechanisms that control cardiovascular development. The overall goal of my research is to create a better understanding in the investigation of molecular mechanisms in heart development. In creating a transgenic zebrafish the key technique is the microinjection of a specific gene with an attached fluorescent destination vector, from a certain species, into its single cell fertilized embryo. This will lead to the visualization of temporal and spatial gene expression patterns as seen by the fluorescence while using non-invasive techniques. The specific aim of the study is to characterize the regulatory enhancer sequence of GATA4, which is known to contribute to heart development. The study will better clarify the specific timing and anatomical contributions of these genes to the structure of the heart.
 * // Overview //**

The genetic model system used in the study of GATA4 is Danio rerio, commonly known as zebra fish. To begin the lab process, I isolated zebrafish DNA by anesthetizing several zebra fish in 200mg/L tricaine. I waited until the fish displayed symptoms of complete sedation, and then sacrificed the fish via scalpel decapitation and then sectioning it into small pieces. Next the collection of zebrafish tissue was snap frozen in liquid nitrogen and pulverized in a mortar and pestle precooled at -800C. Next approximately 1g of the pulverized tissue was add to 7.5 ml lysis buffer in a 50ml polypropylene conical tube. The tissue submerged and was then incubated for 45-60min at room temperature on a rocking platform. The contents of the lysis buffer were transferred to a separate conical tube containing 18ml of 190 proof molecular biology grade ethanol (EtOH). The DNA formed a white gelatinous mass and was recovered by slowly stirring the two layers of lysis solution and ethanol and removed from the solution using a shepherds crook. The DNA was transferred to a fresh polypropylene tube containing 1ml of TE (ph 8.0) allowing the DNA to rehydrate while stored at 40C.
 * // Genomic DNA Isolation //**

The next step involved the selection of the putative promoter sequence for GATA4. The promoter of a gene is the genomic sequence near the transcription start site that modulates the expression of the adjacent gene in response to environmental conditions. The proximal-promoter region can typically be found ~1000 bp up-stream of the transcriptional start site and ~300 bp downstream. The proximal promoter is responsible for containing regulatory elements that direct expression to specific tissues at specific times during development. The putative promoter, or conserved non-coding sequence (CNS) was found using VISTA browser http://genome.lbl.gov/vista/index.shtml. The VISTA browser provided a powerful tool for comparing genomic sequences. I used this software to compare the zebra fish and human genome, specifically searching for homology between the two for the GATA4 gene. I searched for and selected a red highlighted CNS and recorded its position. I then used the University of California– Santa Cruz website: http://genome.ucsc.edu to obtain the actual zebra fish genomic DNA sequence for the highlighted region. Next I designed the sense and antisense primers using Beacon Designer 4.0 by Premier Biosoft International. The primer sequence was confirmed using //in silico// PCR, to generate a precise amplicon sequence. Before the primers were ordered Dr. Balza assisted my design by adding attB sites for gateway cloning to each of the primers. The final product of the primers was order from Integrated DNA Technologies, INC. The primers were freez dried for stability at room temperature. The table below displays the important information obtained from the primer design.
 * // Primer Design //**


 * || Sequene || Position || Length(bp) || Tm0C || GC% ||
 * Sense- Forward || ACCTGGCTGTATCTGGCATT || 162 || 20 || 59.58 || 50 ||
 * Antisense Reverse || TCAGAATCCCTCAGGAAAGAA || 330 || 21 || 58.85 || 42.86 ||
 * Product ||  ||   || 169 ||   ||   ||

Developed by Kary Mullis, Polymerase Chain Reaction is used to produce a relatively large amount of a specific piece of DNA from a small amount of nonspecific DNA. In this case the CNS region of DNA that was selected for in the primer design will be amplified, and the oligonucleotide primers that were designed will serve as the start point for the DNA replication. Beginning with creation of a 100μM stock solution of each pimer, the oligonucleotide primers were spun down and molecular biology grade water was added. A 10μM solution was then made by adding 10μL of each primer solution to 80μL of molecular biology grade water. The working stock solution for PCR was made by adding 21μL of molecular biology grade water, 2.5μL of 10x Accuprime polymerase buffer II, 0.5 μL Genomic DNA in TE, 0.5 μL Primer mix solution and 0.5μL of Accuprime Taq polymerase. Four microfuge tubes were used containing this working stock solution, the tubes were placed in Bio-Rad thermocycler to run the PCR program. The first cycle involved three steps and ran a total of 35 times. The first step raised the temperature to 95°C to denature the DNA, the second step was run at 59, 58, 56.5, and 54°C. for 45 seconds to attach the primers to the DNA. The third step ran at 72°C for 3 minutes to allow the polymerase to copy the conserved non coding sequence. The second cycle continued to run at 72° C to ensure the full extension of the amplicon sequence. And the final cycle lowered the temperature to 4°C to inhibit the amplicon sequence from degrading. I then created a 2.0% agarose gel containing GelGreen DNA stain by dissolving 1g of agarose in 50mL 1x TAE and heating it in the microwave. 2.5μL of GelGreen DNA stain was added to the liquid contents, the gel was poured into a mold and a comb was place at one end. Once the gel solidified, the comb was removed and the gel was barely submerged in 1x TAE buffer to maximize the rate of electrophoresis. 5μL of 1Kb DNA ladder was added in the first lane of the gel and a combination of 1μL 6x Tritrack loading dye and 5μL of the DNA solution from each PCR tube was added to the next several lanes. The gel was electrophoresed for approximately one hour at 110 volts. The DNA was observed, photographed and then extracted from the gel using the Qiaex Gel Extractoin Kit.
 * // Amplification and Isolation of Enhancer Using PCR & Agarose Gel Extraction //**

After the extraction of the DNA was complete. I began the process of cloning the CNS amplicon into an entry vector. 7.5 µL of the attP PCR product, .5 µL of pDONR 221, and 2 µL of BP Clonase II were added to a micro centrifuge tube and incubated at 25ºC for 1-2 hours. After the incubation 1μL of the BP clonase reaction product was added to 20μL of thawed TOP10 //E.coli// cells and incubated on ice for 30 minutes. The contents were then heat shocked for 30 seconds at 42°C and then placed back on ice for 2 minutes. Heat shocking the cells weakened the membrane allowing the DNA to be transformed into the cells. 250 μL of S.O.C. medium was added to the cells and they were placed on a 225 rpm shaker for 1 hour in the warm room at 37°C. Following the incubation and mixing the 200μL of transformed cells were placed on prewarmed kanamycin plates. The plates were inverted and the bacteria was allowed to grow overnight. The plate yielded a colony of TOP10 E.coli that contained the product of the BP clonase reaction. The colony was then picked from the plates and grown up in 15ml conical tube containing 5ml of LB broth and 50 μL of kanamycin sulfate diluted from 100x stock. After approximately 24 hours the conical tube appeared cloudy, indicating sufficient bacterial growth, which was then stored at 4°C. To continue the process the bacteria containing the product of the BP reaction was cryogenically frozen by combining 1.7ml of E.coli in LB media with .3 ml of 80% glycerol and stored at -80°C.
 * // Direct Cloning of the CNS amplicon into an entry vector using BP clonase //**

The QIAprep Spin Miniprep Kit by Qiagen, was used to lyse the promoter insert and the plasmid was isolated for analysis. We were unsuccessful in determining the correct length of the CNS therefore the process was stopped.
 * // Subcloning the CNS into an expression vextor using LR clonase //**

Morpholinos are ~25bp oligonucleotides that are designed to hybridize to the translation initiation site (5’UTR) of target mRNAs to sterically block ribosome-mediated translation. The morpholino backbone is chemically composed of a ring that is thought to confer resistance to nuclease degradation, allowing the morpholino to block translation for as long as seven days after embryo injection. Two wild type females and one wild type male were bread to produce embryos. Injection needles were prepared using the needle puller and then the microinjection apparatus was assembled. Negative pressure was applied on the needle to draw in the CX43 morpholino working solution with phenol red indicator dye present to distinguish the successful injections. Injections into 1-8 cell stage embryos were done with a pneumatic injection apparatus, and the injections were made into the yolk of 40 embryos rather than the cell cytoplasm. The embryos were then separated into a control and morpholino groups in egg water filled petri dishes, and allowed to develop for 48 hrs until they were observed.
 * //Microinjection of Morpholino//**


 * __Results__**

The lab began with the isolation of genomic DNA from zebra fish. The protocol was followed and material was generated from the isolation process. When the DNA was run through a gel no bands appeared in the gel that indicated that we were unsuccessful in the isolation of DNA. Genomic DNA that was provided by Dr. Balza was then used to continue the process of producing the transgenic fish.
 * //Genomic DNA Isolation//**

The GATA4 primers that were selected and designed using computer software were ordered and used in the Polymerase Chain Reaction to produce a large amount of the DNA sequence selected with the designed primers attached. After the preparation of solution and BIO-RAD thermocylcing the contents of the PCR were run through a gel to determine success of the reaction. Figure 1 below displays the resulting gel electrophoresis that illustrates the 1Kb bp DNA ladder in lane one, and and 100bp ladder in lane two. The other two bands are from the PCR reaction coming from the annealing temperature lanes of 59°C and 58°C. These two bands were then sectioned appropriately and isolated from the gel using the QiaexII Gel Extractoin Kit.
 * //Primer Design / PCR//**

The BP reaction was conducted using the extracted DNA from the PCR reaction in the effort to clone the putative enhancer sequence into the pDONR 221-plasmid vector. The BP reaction was conducted and then the heat shock transformation was carried out to add the DNA into the weakend cells of the E. coli bacteria. The results were placed on kanamycin plates and the bacteria grown. I attempted to complete this reaction, transformation, and growth 5 times with my own materials, however I was unsuccessful. To complete this portion of the lab research process I team with Dane Perlick and Lawrence Seymour. By using Lawrence’s materials we were successful in growing a colony of bacteria.
 * //BP Reaction//**

The Morpholino CX43 was injected into 40 wild type zebrafish embryos after 48 hours of development 15 growing fish were observed in comparison to the control zebrafish. Figure 2 below demonstrates the comparision of uninjected control zebra fish on the left and the altered phenotype displayed in the CX-43 morpholinos on the right. The phenotype from the injected embryos shows a curvature in the vertebrate and abnormal development in the areas surrounding the heart confirming the gene knock down.
 * //Morpholino//**

The purpose of this research was to create a transgenic zebrafish to further the understanding of the GATA4 gene in zebrafish heart development. The complete understanding of the GATA4 gene in the development of cardiac myocytes and atrial valves could be very useful in understanding heart development and cause of defects in human heart development because GATA4 is a homologous gene in both humans and zebrafish. In my efforts I was unsuccessful in producing the final product of a transgenic zebrafish. However I did complete several of the steps leading up to the final product and stopped my work in attempting to complete the LR reaction. The lab work began with learning the techniques of isolating genomic DNA from zebrafish. My group for this portion of the lab was unsuccessful in acquiring a successful isolation of the DNA. The error in our efforts occurred when 18ml of 190 proof molecular biology grade ethanol (EtOH) was added to the lysis solution. The ethanol was added too quickly which stirred up the gelatinous mass of DNA and we were unable to recover a substantial amount from the solution. I was successful in my first PCR and this was confirmed by the gel electrophoresis. However I was unable to properly separate the DNA from the gel in my first trial. I then ran the PCR again and this time completed the separation successfully. Figure 1. above displays the results of my PCR product after gel electrophoresis successfully produced DNA is present in the third and fourth lane. The annealing temperature used in the successful reactions was 59°C and 58°C. The BP reaction, next in the process, gave me many difficulties and frustrations. I attempted the BP reaction 5 times until I was completely out of PCR separated product. After unsuccessfully creating bacterial colonies in two different trials I decided to go back and run a gel to determine if I had successfully separated the DNA from gel after the PCR. When this was done a faint band was seen that gave evidence that DNA may be present however after discussion with both Dr.Balza and Dane Perlick I believe I may have made an error in the drying and washing process in the DNA separation from the electrophoresis gel. Dane explained that it was crucial to allow the pellet substance to dry to a white color, I did not allow for the complete drying which would have added the alcohol chemical into my DNA causing it to be damaged and denatured. One of the days in which I was working on the BP clonase the cryo-cooler containing the BP clonase enzymes was left on the table for part of the afternoon. I believe that this occurrence may have damaged the components resulting in a failed product of the BP reaction. Eventually I successfully completed the BP reaction but not until I teamed with Lawrence Seymour and Dane Perlick. In completing the reaction and growing colonies Lawrence’s materials were used and we successfully cultured and later grew up one colony of //E.coli.// The creation of the transgenic fish was halted when Lawrence, Dane, and I were unable to complete the LR reaction. We were unable to grow bacteria on the ampicillin plates, because of incorrect in the isolation of the entry clone plasmid DNA that then inhibited the determination of the correct CNS length. To finalize our lab work Lawrence, Dane, and I bred two wild type female fish with one wild type male to produce embryos. These embryos were then injected with a solution of CX-43 morpholino and a phenol red indicator so that the injection could be visualized. After 48 hours of growth the phenotype of the control and injected zebrafish were analyzed. As suspected the gene knockdown technique was successful and we were able to visualize a curvature in the vertebrate of the zebrafish and also mutations in the area of the heart. Although unsuccessful in my efforts to produce a transgenic fish I believe that with further experimentation and time I would be able to complete the creation of a transgenic zebrafish with the transgene GATA4 being fluoresced at different times during development.
 * __Discussion__**

The goal of producing a transgenic zebrafish was not completed. The process of creating the transgenic zebrafish was extensive and required attention to detail. With continued experimentation and trials it is possible to complete the LR reaction and injection of the transgene into zebrafish embryos. The results of my research will not contribute to the understanding of heart development and expression of the GATA4 gene. However I was able to obtain a greater understanding and appreciation of the process and important procedures involved in genetic research. If I was successful the results of the research could be extremely beneficial in contributing to the expression pattern of GATA4 in zebrafish heart development and how it correlates to human. Researches of this type will be continually used because of the limitless benefits that it offers the field of genetics.
 * __Conclusion__**