Perlick,+Dane

Zebrafish genetics- The GATA 5 Gene.

**PURPOSE**


 * Lab 1**

The goal of this lab is to 1) provide an overview of the zebrafish transgenesis strategy

that we will employ this semester; 2) isolate

genomic DNA from zebrafish (Danio rerio);

3) identify putative enhancer sequences using online bioinformatics tools; 4) design

primer sequences, and 5) familiarize you

with online tools for researching specific

genes and/or genetic disease.


 * Lab 2**

The goal of this lab is to characterize the quantity and quality of the zebrafish genomic DNA isolated in Lab 1.


 * Lab 3**

The goal of this lab is to: 1) Dilute your lyophilized primer oligonucleotides to a suitable storage concentration. 2) Design and execute a PCR reaction to amplify the

proximal promoter region of your assigned gene using zebrafish genomic DNA and

the primers that you designed previous labs. 3) Characterize the amplicon product

of the PCR reaction from lab by agarose gel electrophoresis. 4) Extract the specific

amplicon from the agarose gel using a modified Vogelstein and Gillespie extraction

1


 * .Lab 4**

The goal of this lab is to: 1) quantify the amount of putative enhancer amplicon purified from the gel in the previous lab; 2) direct cloning of the putative enhancer amplicon into an entry vector using BP clonase (see Figure 1); and 3) transform competent E. coli with the BP reaction product


 * Lab 5**

The goal of this lab is to: 1) select two or four (use even number) bacterial colonies

containing the BP reaction product and grow up small overnight cultures, 2) isolate

the entry clone containing your CNS, 3) confirm the correct size of the zebrafish

CNS by performing a restriction enzyme digest, and 4) subclone the zebrafish CNS

into the destination vector (pDes1-01, pDes1-02, or pDes1-03) using LR clonase.


 * Lab 6**

The goal of this lab is to: 1) obtain single-cell zebrafish embryos, 2) microinject the

embryos with the appropriate reporter transgene, and 3) confirmation of transgene

reporter expression.

The goal of this lab is to: 1) obtain FLI1-GFP transgenic single-cell zebrafish embryos; 2) microinject the embryos with morpholinos to inhibit translation of FOG1 and
 * Lab 7**

CX43; 3) confirm successful inhibition of FOG1 (see Walton, 2006) by observing severe pericardial effusion and hypoplastic ventricular wall; and 4) characterize the

unknown vascular phenotype of CX43 morpholino knockdown.


 * Lab 1: Overview of transgenesis strategy, genomic DNA isolation, and bioinformatics**

[|Lab 1] [|Lab 2]

The CNS I choose for this lab it the biggest red peak to the left of the gata 5 gene. (chr23:7,105,455-7,106,165)

3. Identification of conserved non-coding (putative enhancer) sequences

[|1. Compare the zebrafish and human sequence homology for your gene of interest.]

[| 2. Obtain the DNA sequence for each CN]

TTATATGTTCTCTCCGTGTTTGCGTGGGTTTCCTCCGGGTGCTCCGGTTT CCCACACAGTCCAAAGACATGTGGTATTGGTGAATTGGATAAGCTAAAAT TGATCGTAGTGTATGAGTATGAATAAGTATGTATGGATGTTTCCCAGTGA TGGGTTGTAGCTGAAAAGAAAATGTATGAATGAATAATTAAACTACATAC TGTACCTTTTTGCTCCTGGC[ AGCTGTATCATATCCTACTGATCCTGACAG ACGGGGTGGTCACAGACATGGCGGACACACGGGAGGCGATTGTCCGGGCC TCATACCAGCCCATGTCCATCATCATAGTGGGAGTGGGAAACGCAGACTT CACAGACATGCAGATCCTCGATGGAGACGACGGGGTCCTGCGCTCACCCA AAGGAGAGCCCGTCCTGAGGGACATCGTCCAGTTTGTGCCCTTCAG ]AGAC TTTAAAACGGTCAGTCTTTCTCTGTCATTTGGTATTTTACTCAGTTTCTC AGAATTATGAATGTAGTGTTTGTGTAAAAGAAATGTTTGATTGTTTTACT ACTAATGATATTTCTTCCAAATTAAGATATATATATATATATTAGGGATG TAACAGTATCAGAATTTCATGGTACAGTAATACCTCGGTATGAATGTCAC GGTAAGGTATTTATTGAATCATTTACAGGAAAAACAAAACTTATGAAAAT ACTCCAAAAAA

CNS AGCTGTATCATATCCTACTGATCCTGACAGACGGGGTGGTCACAGACATG GCGGACACACGGGAGGCGATTGTCCGGGCCTCATACCAGCCCATGTCCAT CATCATAGTGGGAGTGGGAAACGCAGACTTCACAGACATGCAGATCCTCG ATGGAGACGACGGGGTCCTGCGCTCACCCAAAGGAGAGCCCGTCCTGAGG GACATCGTCCAGTTTGTGCCCTTCAG

[|Primer 3 Output]

OLIGO [|start] [| len] [| tm] [| gc%] [| any] [| 3'] [|seq] LEFT __ [|PRIMER] __ 196 23 58.03 43.48 5.00 1.00 CATACTGTACCTTTTTGCTCCTG RIGHT PRIMER 482 22 57.05 40.91 3.00 1.00 CCAAATGACAGAGAAAGACTGA SEQUENCE SIZE: 711 INCLUDED REGION SIZE: 711

LP TM = 58.03 RP TM= 57.05

[|UCSC In-Silico PCR Results]

287bp

CATACTGTACCTTTTTGCTCCTGCCAAATGACAGAGAAAGACTGACATACTGTACCTTT TTGCTCCTGgc[ agctgtatcatatcctactgatcctgacagacggggtggtcacagac atggcggacacacgggaggcgattgtccgggcctcataccagcccatgtccatcatcat agtgggagtgggaaacgcagacttcacagacatgcagatcctcgatggagacgacgggg tcctgcgctcacccaaaggagagcccgtcctgagggacatcgtccagtttgtgcccttc ag ]agactttaaaacggTCAGTCTTTCTCTGTCATTTGG

[|Lab 3] I first diluted the oligonucleotide primer to a 10 micro mole primer solution. Then I started the PCR reaction. I set up four reactions and conducted them at different temperatures by placing them in differnt rows in the machine. I followed the handout for the processed above.
 * Lab 3 PCR reaction 9-19-2011 **

PCR TEMPS

PCR 1 A-58.6 B-57.2 C-56.2 D-55.5

PCR 2

1-59.0 2-58.5 3-57.5 4-55.8 5-53.5 6-51.9 7-50.7 8- 50.0

+ Control 1-58.5 2-57.5 3-55.8 4-53.5

After the reaction was complete, I transferred my reactions into the freezer for storage.

I ran a gel of a my PCR reaction.

Cut out the piece of gel in the 8 lane that contained dna that was aproximently 300 base pairs long. I placed it in a centerfuge tube that weighed 1.11g.




 * Lab 4 BP cloning**

[|LAB 4]

**Methods **  In the process of creating a transgenic zebra fish (Danio rerio), microinjection is the principal procedure. It involves the injection of a specific gene that has an attached fluorescent destination vector into a fertilized single cell embryo. This will lead to the gene being expressed as fluorescence in different organs as the organism develops. To begin the transgenesis process, I isolated the zebrafish DNA by anesthetizing it in 200mg/L tricaine until it was completely sedated. I then sectioned the zebra fish into small pieces and snap froze the tissue in liquid nitrogen and pulverized it into a powder using a pre-cooled mortar and pestle. 1 gram of the powder was added to a lysis buffer and submerged. This lysis buffer was than added to a clean 50 ml conical tube containing 18ml of 190 proof molecular biology grade ethanol. Using a Shepherds crook, the DNA was recovered and added to a polypropylene tube containing 1ml of TE Buffer that allowed the DNA to rehydrate. The tube was then stored at 4 ºC This step involved the discovery of the putative promoter sequences. The promoter of the gene is the sequence of DNA close to the transcriptional start site that alters the expression of the gene in response to different environmental stimuli. The putative promoter, or conserved non-coding sequence, was found via VISTA browser ([]), and the DNA sequence of the CNS was obtained by the University of California– Santa Cruz website: [|http://genome.ucsc.edu]. The CNS I choose for this lab it the biggest red peak to the left of the gata5 gene. (chr23:7,105,455-7,106,165) I then designed a sense and antisense primer using Primer3 input (version 0.4.0). The following table includes important information obtained for the remaining steps. PCR or polymerase chain reaction is a process to form a large amount of a specific piece of DNA from a small amount of nonspecific DNA. In order to prepare a primer stock solution, the oligonucleotide primers were spun down and molecular biology grade water was added to create a 100μM solution. Then a 10μM solution was prepared by adding 10μL of each primer solution to 80μL of molecular biology grade water. A working stock solution for PCR was then made by adding 21µL of molecular biology grade water, 2.5µL of 10x Accuprime polymerase buffer2, .5 µL of genomic DNA in TE (DNA provided from Dr Balza), .5 µL of the primmer working stock solution, and .5 µL of accuprime Taq polymerase. I then ran a PCR program using a Bio-Rad thermocycler. I ran a PCR at temperatures of 59.0, 58.5, 57.5, 55.8, 53.5, 51.9, 50.7, and 50.0 degrease Celsius. In order to determine if the PCR worked correctly, I ran a 2.0% aragose gel containing gel star. I ran lanes containing 5 µL  each of my PCR products combined with 1 µL of 6x tritrack loading die. I also ran a lane with 5 µL of a  100bp DNA ladder to determine the size of the PCR product. <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">. The gel was then electrophoresed for 1 hour at 100 volts.
 * //<span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Overview //**
 * //<span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Genomic DNA Isolation //**
 * //<span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Primer Design //**
 * || <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">Primer Sequence || <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">Position  || <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">Length (bp)  || <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">TM (ºC)  || <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">GC%  ||
 * <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">Sense || <span style="background-color: white; display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">CATACTGTACCTTTTTGCTCCTG  || <span style="background-color: white; display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">196  || <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">23  || <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">58.03  || <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">43.48  ||
 * <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">Antisense || <span style="background-color: white; display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">CCAAATGACAGAGAAAGACTGA  || <span style="background-color: white; display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">482  || <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">22  || <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">57.05  || <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">40.91  ||
 * <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">Amplicon ||   ||   || <span style="display: block; font-family: 'Times New Roman',serif; font-size: 12pt; text-align: center;">286  ||   ||   ||
 * //<span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Amplification of Proximal Promoter using PCR and Agarose Gel Extraction //**


 * //<span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Direct cloning of the CNS amplicon into an entry vector using BP clonase //**

<span style="font-family: 'Times New Roman',serif; font-size: 12pt;">After the DNA was extracted from the gel, 7.5 µL of the PCR product, 2 µL of BP Clonase II, and 0.5 µL of pDONR 221 were added to a microcentrifudge tube and incubated at 25ºC for one and a half hours. Then 1 µL of the BP clonase reaction product

<span style="font-family: 'Times New Roman',serif; font-size: 16px; line-height: 24px;">combined with 20 µL of thawed TOP10™ //<span style="font-family: 'Times New Roman',serif; font-size: 16px; line-height: 24px;">E.coli //<span style="font-family: 'Times New Roman',serif; font-size: 16px; line-height: 24px;"> and incubated for 30 minutes on ice //<span style="font-family: 'Times New Roman',serif; font-size: 16px; line-height: 24px;">. //<span style="font-family: 'Times New Roman',serif; font-size: 16px; line-height: 24px;"> The mixture was then heat-shocked for 30 seconds at 42ºC and then cooled back down on ice for 2 minutes. For the cells to recover, they were shaken for 1 hour at 225 rpm at 37ºC. Then the cells were plated on a prewarmed kanamycin plates. The plates were then inverted and placed at 37ºC to incubate for 24 hours. After attempting the BP clonase reaction 5 times, I was not able to obtain bacteria. This will be further discussed later. <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Teaming up with classmates Lawrence Seymour and Tyler Schoen, we were able to obtain one bacterial colony. The colony was picked from the plate and grown up in a 15mL conical tube containing 5mL of LB broth and 50µL of Kanamycin sulfate for another 24 hours. The conical tube became cloudy once there was sufficient bacterial growth and was stored at 4ºC.


 * //<span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Subcloning the CNS into an expression vector using LR clonase. //**

<span style="font-family: 'Times New Roman',serif; font-size: 12pt;">For the LR reaction I used the QIAprep Spin Miniprep Kit by Qiagen. The bacterium was lysed and their plasmids were purified for further investigation. We attempted to confirm that we obtained the correct size of the CNS. We were unsuccessful in completing the LR reaction; therefore, the experiment was stopped there.


 * //<span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Microinjection of Morpholino in Zebra Fish //**

<span style="font-family: 'Times New Roman',serif; font-size: 12pt;"> First, wild-type zebra fish were mated and their eggs were obtained. Negative pressure was applied to draw in the FOG1 translation-blocking morpholino working solution. A pneumatic injection apparatus was used for the injections. 5-7nl injections were made into around 40 single cell embryos. The injected embryos were then placed in egg water and incubated until further analysis.

**<span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Results: ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Morholino results: <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">vfd

<span style="font-family: 'Times New Roman',serif;">