Seymour,+Lawrence

Zebrafish Genetics

September 1, 2011

Selected wild-type zebrafish, and anesthetized it in a diluted solution of tricaine. Once the zebrafish was anesthetized, it was sacrificed via scapel decapitation. The head, tail, and any other large bony parts were removed. The rest of the fish was sliced into small pieces. The small pieces were then placed into a mortar, and liquid nitrogen was poured over it to "snap-freeze" it. Once it was frozen, the zebrafish was ground into a fine powder. 7.5 mL of lysis buffer was placed in a tube along with the powdered zebrafish. The solution was then placed on a rocking platform, to allow the zebrafish powder to settle.

I switched my gene from SRF to gene ISL1.

September 6, 2011

Weighed out approximately .25g of agarose, and mixed with 50mL of 1x TAE. This solution was microwaved for 60 seconds, until the agarose was completely dissolved. 2.5 microliters of GelGreen was added to the solution and mixed together. We then poured the gel and waited for it to set. After it was set, three samples were injected into the wells of the gel. In the first lane, we put a high range DNA ladder. In the 3rd lane, a combination of Tri-Track and control DNA. In the 5th lane was a combination of Tri-Track and our DNA that we isolated from the zebrafish.

September 8, 2011



>danRer4_dna range=chr5:53127992-53129232 5'pad=0 3'pad=0 strand=+ repeatMasking=none CATCTAGATTTTTACAGTGGCCAAGCACATCTGTGCAGTGTTTATAGTTG TAGGGAGATTATGTATTATTTGTATATGCAAAAAAAGCGAAGATAAAAGC TTATAATTCAATTAAATAGGTTTCAACGTAAAAAATAATACTGCACTTTA TCCGTTTAGTTATCTGCCTGTGAATCAGATAGCTTTATGCTTTAAAGGAA CACCAAATACAAATTCCCAACAGTAATCAGATATTTCTAAAAGAGTAGAA CAACAGAAGTGTCGTCAAAGCAAGGGAGTGTCGTGACTTTTTATTTCTCT TTTTGCATTTGATGCCTAGGCCCACTCCTTTGGGAGATGAAACGAAAACT CTGTTATAAAATCATGAAAAGGATATGGACAACAGCAGGTGGGCAAATCT ATCAAAACCCCTGGCAAACGCACATGCAAGCGTACACACATAAAGGGGCA AA [ATCATTTTAATTAGCTGAGTGAATGTGATTTGCTGAATGCGGGGAACT AGGCTCTGCACACATTAAAATTGGTCTAATTTTCTGCAAAAAAGTCCCAT CTGAGTGGACCTGGCCACAGTCAATCAAGTTAAAAGCTATGGGTGCTTAA TTTGATTTACCAATATAAAATGCAAATGAGGTGATTAAGTGGAGAGGGGA GGCAGAGTAGGAGCCTCTTTTAAACCATCAAGTTAAATGTGAACAGACAT CGGACTGGCAGCAGCAAGAATGTTTTAGCATATTCGTTTGATTAGAGGTA CAAAAATTTAATTAGTGTGGCTAATTGCTTGACAAATTGCAGCACACTAC TGAAAAGACAGATTTTTTTTTTAAAACCGTGCAAAACCCCCTCCGTGTGG AAATTTTGTCCAAATGGCCCCTATGCCAATATGTGAAAAGCATAATTAAA TAAATGGAAGATGGCACAACAGTACCTTACAATAGCAAATGAGATAATGC CTGTAATTAGGTGGGACACAAGTCTATGTCCATATGTCGTGTTTCTC] TTC AGCTACTCTCCGTTCCTCTCGTAGGACAAATCTAATAAGCCTTCTTAACT AGGTGAGGCTCTCCTGGGATGCTGAAGAGCTGCTGGTCTGAGTCGTCATA TAGAGGAGCAGTAATCGGAGTATGAAGCATATAATCAGGGCAAGCTCCTG TACTACAACAAACATGGTGGATTTTATGCTAGAGAAGTAGTGCATTTATA ATAGTATTCTGTGATAAATTCTGTTTTTTTTTTTTTCAAAT

OLIGO [|start] [| len] [| tm] [| gc%] [| any] [| 3'] [|seq] LEFT PRIMER 421 20 60.39 50.00 4.00 0.00 CACATGCAAGCGTACACACA RIGHT PRIMER 1020 22 60.15 54.55 4.00 2.00 GAGAGGAACGGAGAGTAGCTGA SEQUENCE SIZE: 1241 INCLUDED REGION SIZE: 1241





After trying the primers in the online PCR, it appears as if the primers will only bind at the designated area.

Lab 3 PCR Reaction 9-19-2011

https://balzalabnotebook.wikispaces.com/file/view/Lab+3+PCR+%26+Amplicon+purification.pdf

Following the lab protocol outlined above, I ran a PCR at four different temperatures:58.6, 57.2, 56.2, 55.5 (all temperatures Celsius) when the PCR products were run through a gel, I was unsuccessful. Therefore I ran the PCR again at 8 different temperatures to maximize the chance of obtaining a good product.

59.0 58.5 57.5 55.8 These were the temperatures at which we ran the PCR. Only the second and fourth PCR reactions were successful 53.5 when the gel was ran. 51.9 50.7 50.0

This was also done when I ran a control to ensure that I was not messing up the process. The control was the primers created by Tyler Schoen, and they were run at the 2-5th temperatures of the second attempt.

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 mecha- nisms that control cardiovascular development and remodeling. The overarching goal of this investigation is to elucidate key molecular mechanisms of heart devel- opment and pathophysiology.
 * //1. Overview of transgenesis strategy//**

The visualization of temporal and spatial gene expression patterns is foundational for understanding the role of genes during development. While indirect immunofluo- rescence and //in situ// hybridization techniques provide atemporal snapshots of gene expression at the protein and RNA level respectively, spatial and temporal examina- tion 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. Fur- thermore, conventional techniques do not allow one to isolate specific gene en- hancers 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 differ- entiation of heart muscle cells. While the importance of these genes in heart devel- opment 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 identi- fied 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 limita- tions.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 frequen- cy 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


 * //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 revolu- tionized 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 contrib- uted 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 trans- lation 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). The morpholino ring is thought to confer resistance to nuclease degredation, allow- ing the morpholino to block translation effectively for as long as seven days after embryo injection (see Figure 2).
 * //Introduction to gene knockdown using morpholinos//**

Methods The goal of this experiment is to inject zebrafish (Danio rerio) with our desired transgene that has been combined with a transgenic fluorescent reporter gene such as green fluorescent protein (GFP). With keen observation and diligence, the reporter gene would glow where ever the transgene is expressed. This allows for the expression patterns of the desired gene to be tracked and noted during the development of the organism
 * //Overview//**:

The first step in the transgenesis experiment is to isolate the desired DNA from the model system. The following protocol was used to obtain the genomic DNA from the zebrafish. One zebrafish was anesthetized using 200mg/L tricaine until the fish was completely sedated. The model was then sacrificed and cut transversely into small pieces. Liquid nitrogen was used to “snap” freeze the zebrafish tissue, after which the tissue was pulverized in a pre-cooled mortar and pestle. After the liquid nitrogen had evaporated, approximately 1g of the powdered tissue was added to 7.5 mL of lysis buffer, and submerged. Incubate solution for 45-60 min at room temperature on a rocking platform. The solution was then transfered to a separate conical tube containing 18 mL of 190 proof molecular biology grade ethanol. Recover the DNA by stirring the two liquids together with a Shepard’s crook at which point the DNA should adhere to the crook. Remove the crook and wash the DNA again in a fresh tube containing 5mL of EtOH. Transfer the DNA to a fresh tube containing 1mL of TE buffer, and allow the DNA to rehydrate at 4 degrees C.
 * //Isolation of zebrafish genomic DNA//**

The second step in the process was to identify a non-coding (putative enhancer) sequence that would serve as a potential base for the experiment. The promoter of the gene is the sequence near the transcriptional start site that adjusts the expression of the related gene in accordance to different environment stimuli. The CNS or conserved non-coding sequence was located using the VISTA browser ([|__http://genome.lbl.gov/vista/index.shtml__]). The DNA of the sequence was obtained through the University of California-Santa Cruz website: [|__http://genome.ucsc.edu__]. The sense and antisense primers were designed using Primer3 input (version 0.4.0). The primers were confirmed using the //in silico// PCR device on the UCSC genome browser. This is done to ensure that the primers will only attach in one place on the genome.
 * //Primer design//**

PCR or polymerase chain reaction is an important tool for a geneticist. It allows for the geneticist to produce large amounts of specific pieces of DNA from a small amount of nonspecific DNA. This greatly lowers cost, and is much less time consuming than previous methods. When working with PCR, the primers must be prepared from their shipped state. The tubes containing the primers were spun down and 100 μM was created. This is done by multiplying the number of moles of primer in the tube by 10 and adding that much μL water to the tube. To create a 10 μM working solution, 10 μL of the sense primer and 10μL of the antisense primer were added to 80μL of molecular biology grade water. The working solution is stored at -20 ℃. The PCR solutions contain 25μL solutions in 0.2mL PCR tubes. They contain: 21.0 μL molecular biology grade water, 2.5 μL 10x Accuprime polymerase buffer II, 0.5 μL genomic DNA in TE, 0.5 μL primer working solution, and 0.5 μL Accuprime //Taq// polymerase. The PCR reaction was then run using the Bio-Rad Thermocycler in the Wisconsin Lutheran College Genetics Lab (S248). After the reaction was run, store the product at -20℃ until ready to proceed. To check if the PCR reaction was successful, the products are run through an agarose gel and compared to a DNA ladder to check if the correct DNA products are present. The agarose gel is prepared using 50mL 1xTAE, and then dissolved using a microwave. The amount of agarose to add depends on the desired concentration of the solution. 2.5μL of GelStar DNA stain was added to the solution. The gel was run with 5μL of a DNA ladder, and 5μL of the PCR product which had already been combined with 1μL of TriTrack loading dye. The gels would take approximately 1 hour to complete. In order to separate the DNA from the gel, it is necessary to purify the DNA out. First the DNA is sliced out of the gel, and then it is weighed. Once it is weighed, the gel can be purified using the Qiaex II Gel Extraction Kit. The product is stored at -20℃ until further use.
 * //Amplification and isolation of enhancer using PCR & agarose gel extraction//**

Following the gel extraction, the following elements were added to a 1.5 mL micro-centrifuge tube at room temperature: 7.5 μL PCR product, 0.5 μL pDONR 221, and 2 μL BP Clonase II enzyme/buffer mix. The solution is briefly vortexed and then incubated at 25℃ for 1-2 hours. 1 μL of 2 μg/μL Proteinase K solution was added and this solution was incubated at 37℃ for 10 minutes. 1 μL of the BP clonase reaction was added to a micro-centrifuge tube and placed on ice to chill. Meanwhile a vial of TOP10 //E.coli// cells are thawing. Add 20 μL of the cells to the chilled micro-centrifuge tube. This solution is incubated on ice for 30 minutes. The DNA and the bacteria are combined when the cells are heat-shocked for 30 seconds at 42℃. The cells are then placed back on ice for 2 minutes. 250 μL of S.O.C. Medium is added to the cells aseptically. Shake at 225 rpm (37℃) for 1 hour. Spread 100-200 μL of the cells on a pre-warmed kanamycin plate, inverted, and incubated at 37℃ overnight. The plate should have colonies grown up the following day. The plates can be stored at 4℃ until ready to grow them up.
 * //Direct cloning of the CNS amplicon into an entry vector using BP clonase//**

Transfer colonies to 15mL conical tubes which contain 5mL LB media with kanamycin. Allow to grow overnight. Combine 1.7 mL of //E.coli// in LB media with 0.3 mL of 80% glycerol in a 2 mL cryopreservation tube. Vortex briefly, label well, and store at -80℃.
 * //Preserving E.coli through cryogensis//**

Isolate the entry clone plasmid DNA using the QIAprep Spin Plasmid Miniprep kit by Qiagen. The size of the cloned CNS insert is confirmed by cutting the plasmid with two restriction enzymes, and then running it through a gel. The solution to run through the gel to see if the plasmid is the correct size consists of 18μL water, 2.5μL 10x NEB buffer #4, 2.5μL 10x BSA, 1.0μL plasmid DNA, and 1.0μL Apal enzyme. This mix is incubated at 25℃ for 1 hour, and then EcoRV is added and the temperature is raised to 37℃ for an additional hour. To subclone the CNS, the following components were added to a centrifuge tube: 0.5μL //Entry clone// containing CNS, 0.5μL destination vector, 6.0μL 1xTE buffer. The LR Clonase II Plus enzyme is vortexed briefly and 2μL is added to the above components. Vortex the components and then spin down. Incubate reaction at 25℃ for 16 hours. Add 1μ of 2μg/μL Proteinase K solution and incubate at 37℃ for 10 minutes. The LR reaction needs to be transformed into the //E.coli//. 1μL of the LR reaction is added to a centrifuge tube and placed on ice, while a vial of TOP10 //E.coli// cells are being thawed on ice. Add 20μL of cells to the tube containing the LR reaction. Incubate the tube for 30 minutes. Heat-shock the cells for 30 seconds at 42℃. Place the heat-shocked cells back on ice for 2 minutes. Add 250μL of S.O.C. Medium to the cells and shake at 225 rpm (37℃) for 1 hour. Spread 100-200μL of cells on pre-warmed ampicillin plates, and incubate the inverted plates overnight at 37℃. Store the plates at 4℃ until ready to grow the colonies up.
 * //Subcloning the CNS into an expression vector using LR clonase//**

Pull capillary tube to obtain needle for the injection apparatus. Using negative pressure, the CX43 morpholino is drawn into the needle and is ready for injection. The embryos were lined up against a slide in a petri dish. This allows for the injector to slide the dish and line the next embryo up. The embryos were all in the 1-8 cell stage, and 5-7 nl injections were made into the yoke sac. After injection the embryos were placed in egg water, and allowed to grow for 48 hours to see the phenotype.
 * //Inhibition of mRNA translation by morpholino injection//**

The BP reaction was run several times until a single colony was produced and cultured. After running an LR reaction we were unable to get the LR reaction to grow bacteria, and thus our attempt at obtaining a transgenic fish was terminated here.