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Transcript of DevineThomas_BalciunasZebrafishLab_URP_ResearchProject
Gene Traps using the GAL4-UAS system in Zebrafish:
Inverse PCR (iPCR)Devine Thomas, Darius Balciunas, Jorune Balciuniene
Introduction: GENE TRAPPING
What is gene trapping?
It is a form of insertional mutagenesis specifically designed
to disrupt gene function by producing intragenic integration
events (Evans,1998).
Purpose?
We use it for targeted gene expression, investigation of roles
of certain genes, and gene mapping in zebrafish.
Introduction: GENE TRAPPING(contd)
How does it work?
It involves a gene trapping cassette with a promotorless
reporter gene/genetic marker flanked by an upstream splice
acceptor (SA) and a downstream polyadenylation sequence
(polyA).
The cassette is inserted into an intron of an expressed gene,
where the original promoter of that gene transcribes the
gene trap cassette by splicing in the exons upstream of the
insertion site along with the reporter gene/marker.
Introduction: GENE TRAPPING(contd)
This process gives a “fused” transcript with the gene trap
cassette and reporter gene along with the original exons
upstream of the expressed gene.
- This transcript encodes a nonfunctional cellular protein but also
expresses the reporter gene.
This gene trap inactivates the cellular gene, but reports the
expression of the trapped gene at the insertion site.
- It provides a gene trap sequence tag for a quick identification of the
inactivated gene.
Identifying Gene-traps in genome of Danio rerio
In this lab we have constructed gene traps using the
GAL4-UAS system that can produce transgenic fish
carrying reporter genes for fluorescent proteins
downstream of UAS, when inserted into the genome
(Asakawa & Kawakami, 2009).
Green Fluorescent Protein (GFP) Red Fluorescent Protein (RFP) Blue Fluorescent Protein (BFP)
Markers
Gene Trap, GAL4-VP16: Inverse PCRGene trap Construct with GAL4-VP16:
Gal4 fusion protein
Exon 1 Exon 2 Exon 3BFPTol2 Tol2SA
Activation of the trapped genes’ promoter
Tol2 Tol2RFP
p(A)
p(A) GFP
zp(A)
p(A) UAS
^GAL4-VP16
δ-Cry
δ-Cry
Gene Mapping: Inverse PCR
How do we do that? Inverse PCR (iPCR) & Sequencing
We can use inverse polymerase chain reaction (iPCR) to identify
gene trap insertion sites and also recover genomic sequences
that flank both sides of the transposon vector cassette
(Hermanson et al., 2004).
Involves three steps:
- Digest genomic DNA using restriction enzymes:
N – NlaIII
T - TaqI
X – XmaJI (AvrII), NheI, SpeI, XbaI
B - BamHI,BgIII,BcII
- Ligation of digested vector DNA using Roche Buffer and Ligase.
- Amplification of flanking sequences through PCR twice using outward-
facing primers.
Outline of Inverse PCR process: An example
5’ 3’
5’ 3’
*IR/DR – Direct/Inverted Repeats*RE = Restriction Enzymes
Gene Mapping: Inverse PCR (contd)
^GAL4-VP16
IR/DR (L) IR/DR (R)
Tol2F8 Tol2R3Tol2F10 Tol2R4Digest
IR/DR (L) IR/DR (R)
RE
Restriction Enzymes
RE
Ligate
IR/DR (L) IR/DR (R)
NEXT
RE
RE RERE
Tol2F8Tol2F10 Tol2R4Tol2R3T25’No1
S13’No1
T25’No2
S13’No2
Ligate
Tol2R4Tol2R3Tol2F8Tol2F10 S13’No1
S13’No2
T25’No1
T25’No2
PCR1, PCR2
Using GAL4-UAS system and iPCR
The next step…
Run PCR2 reaction mixes on gel (gel electrophoresis)
Cut out significant DNA bands and send it for sequencing through GeneWiz.−Use Gene Extraction Kit and then samples sent out for
sequencing
Identify and confirm candidate loci.
Use regular PCR to see linkage of gene trap to RFP expression.
What I have done so far...
DNA prep and iPCR using the GAL4-UAS system:
Completed DNA prep with embryos of zebrafish vector lines, both negatives and positives.
digested, ligated and completed iPCR on three different lines of zebrafish containing either the S8 or B6 vector.− S8.1D F1.1 (+ only)−B6. 7B F1.3 (+/-)−S8.4A2 F1.1a (+/-)
Identified candidate loci after getting results from gene sequencing.
Confirmed gene trap was linked to RFP expression through regular PCR.
1st Vector Line: S8.1D F1.1♂(+)S8 Vector
This line is mainly ubiquitous, shows enhanced RFP expression in central nervous system.
Only positives and one single integration identified.
Digestion done: N, T, X, B Gel Electrophoresis Bands:Ligation with Roche Buffer & Ligase 8 PCR tubes for PCR1 &PCR2
PCR1 with primers: Tol2F8 (5’) & Tol2R3 (3’) T25’No.2 (5’) & S13’No.1 (3’)PCR2 with primers: Tol2F10 (5’) & Tol2R4(3’) T25’No.1 (5’) & S13’No.2 (3’)
T3’ T5’cSA ^Gal4-VP16 zp(A)
CryL BFP pA
S8.1D F1.1♂ (+): IntegrationSequencing results:
Integration identified in Exon 2 Gene: Fam100ab
- family with sequence similarity 100, member ab - protein coding gene
GTTCATAACCTTGAATTTGTGAATAAAATCCTTTGCTAGCAAAATTATTAAGTTGGAAAG
AAAATAAATGTTTTCACATTGCTTCTCAAGGGTAATAAGTACAAATAAAACATATTTAAA
GCAAAGCTCAAAACTATCATTTATGATATGTACAATAAACTGGCAGATATCTTGCCAAAA
TCTCCTTGTTTCGACTGTTGACATCACCACGACAGGGCTTATAGGGGTGCGACCAGGTCC Fam100ex2-F1 Primer 1 (forward)CAGCAGAATAAAACAGCCCTTTCTGGAATATCCACACGAATATTAGTCTCCATGTGGGTG
TACATTGTTGTATTATCAATATTATCGTTCACATGGTAAAACCTGTAAGCAGTTTGTGAG
TGTAACTGTACATGAGGTGCTTGCTAAGGGTTAACTGAATTTCATTCGCTTTGCATGTGA
CCGGTAGTACTACTATTTCTGAGTAATCGAGACAAATTCCTTTAAGTTTAGATATCCAAA
TTTAAAAACCCCGACACTAATAAAAAAATACCCGAAGTTTAAACTAGCAAGGCTTTGAAC
AGACAACAATGCGTCCTTAGTGAAGGCGATCTTGTTGTTATTACAGTAGTGTAAACAGCG
CGGTGTCAAGCGCGTGTTCTCTCGGTTTCCTGGAATCGCCCTCCTCCCCCACAACTTTGG
ACACGTTTGCAAAAACCCGACTCCCATGAGTCCAGATTAATTTAACATCTTAAAACAATA
ACATATCTAATTAACCAACAAAGGGCGACATGGTGAGCTAAACAACCAAAGTCGCAATTT
TTCAAACGAAATAAATAAGCTCTGGCTAGAAACACGCCAGCCTGTGTTTATTTATGTCTG
ACTCGTGGTTTCAACATGGCGGAAGTCATCATAACAAATATAAAAAAGGGCCAAAACACA
ATGACTAAAATAACAAGAGTATACTCACCATTTGATGGCTGTAAGGAATATTAGCCTCTT Fam100in2-R1 Primer 2 (reverse)GGAAAAATGTACTGAGGGCAGCCTGTAAGACAATACAATATGGATTATTTGTAATTCAAC
AAATACTCATTTGGAAGTACGCGAATTTAATGAATTTAACATAATATTATTTAATTCGCC
TTATCATAATACTTTCTACCTCATAAATGAATTATTTCAATGTCATATTTGTTTAGATTG
TCTGTTGCAGTGTTTGTTTTGCGTGATTATAAAATATAACACGCTTCAAATAAACAAAGA
GTTAATTGATTTGTGTAAATTTATATTAACGTTATATTAAATGAGATTCTTCTTCATGTT
AAGAGTCCTACCAAACAAACATAAGTTA
S8.1D F1.1♂ (+): PCR confirmation
Regular PCR with Tol2F8 (5’) primer + Forward/Reverse primers
2 PCR tubes, Labels: Gel Bands:
1 = genomic control with F1/R1 primers2 = vector (+) DNA with F1/R1/Tol2F8(5’)
Fam100ex2-F1 Forward PrimerFam100in2-R1 Reverse Primer
Results:
1 band in the genomic column (tube 1)2 bands in the vector column (tube 2)
* Gene trap integration – confirmed* Linkage to RFP - confirmed
2nd Vector Line: B6.7B F1.3 ♀(+/-)
B6 Vector
Fairly ubiquitous line - shows enhanced RFP expression in midbrain. - Positives and negatives
Digestion done: N, T, X, BLigation with Roche Buffer & Ligase 16 PCR tubes for PCR1 & PCR2
Gel Electrophoresis Bands:PCR1 with primers: Tol2F8 (5’) & Tol2R3 (3’) B1’5’No.3 (5’) & B13’No.1 (3’) PCR2 with primers: Tol2F10 (5’) & Tol2R4(3’) B15’No.2 (5’) & B13’No.2 (3’)
T3’ T5’cSA ^Gal4-VP16 zp(A)
CryL BFP pA
B6.7B F1.3 ♀(+/-): IntegrationSequencing results:
Integration in Exon 4
Gene: zmynd8- Protein kinase C-binding protein 1 in humans (considered a transcriptional regulator)
zmynd8 exon 3 frame 4, exon 4 frame 0. >chromosome:Zv9:11:19009235:19010041:-1TTGTCTAAATTTGCCCTCTTCTGTTTTTCCACACCCTGATCGATCACCTTGGTGCCTGTTTTGTATTGTCTTGTAACCTATAATAGCAATGTTTTGAAACCCCGACCCTCTGTTTCTCTCGCCCGCATTCCAGTCAGAACTAAGACATGGTCCCTTTTACTATGTGAAGCAGCCCGCACT
CACCACAGACCCTGTTGATGTTGTACCGCAGGACGGGCGGAATGACTTCTACTGCTGGGT zmyndex4-F1 Primer 1 (forward)GTGCCACCGCGAGGGCCAGGTGCTCTGCTGTGAGCTCTGCCCGCGCGTCTACCACGCCAAGTGCCTCAAACTGGCTGCAGAGCCTGAAGGAGACTGGTTCTGCCCTGAATGTGAGGTACT GCCTCACTTTGTGCTTGAACAGCGCATCATCACTCTGCAGATTGTTAAATTATTCTGCAGTATTTTAACACTCCTTAAATAAAGGTTTTAAATTAACGTTTTTGCTGTGATATCCTAGAAGACTCTACTGCTATATGGGTTCCTCAAAAAATCAATTGTTTAAAGCAATCTGAAGAAACTTTTTCCTCTATTAAGAATCTTTTATGGAATCTAAATGTTTATGGATGTTAAAGGCCCCAT
ACAGAATCGCCAAATCTAAGTTCACCTAATTAATAGTACCCTTATTTTTTACATTTTTAA zmyndin4-R1 Primer 2 (reverse)AGTTATTTCATATTAATACCATATAATAAAAGCAATAAATGTATATATACAGTGTATATGTATATTGTAAAAAAATCCTGGGTTCCACACAATTTCTTTACAAAGCAAAAGTTAACATCATTGTTTTTGATCTCACAAGGAAACCTA
B6.7B F1.3 ♀(+/-): PCR Confirmation
Regular PCR with Tol2R3 (3’) primer + Forward/Reverse primers
5 PCR tubes, Labels: Gel Bands: GC = genomic control with F1/R1 primers1 + = vector (+) DNA with F1/R1 primers1 – = vector (–) DNA with F1/R1 primers2 + = vector (+) DNA with F1/R1/Tol2R3(3’)2 – = vector (–) DNA with F1/R1/Tol2R3(3’)
zmynd8ex4-F1 Forward Primerzmynd8in4-R1 Reverse Primer
Results:
2 bands only in the vector column (tube 4) - mutant band 200 bps larger than genomic band
* Gene trap integration is confirmed.* Linkage to RFP?
3rd Vector Line: S8.4A2 F1.1a ♀(+/-)
S8 vector
This line is neural, shows RFP expression in retina.
Positives and negatives (5’ only) Digestion done: N, T, X, BLigation with Roche Buffer & Ligase 8 PCR tubes for PCR1 & PCR2 (5’ only)
Gel Electrophoresis BandsPCR1 with primers: Tol2F8 (5’) T25’No.2 (5’)PCR2 with primers: Tol2F10 (5’) T25’No.1 (5’)
*3’ sequencing produced no bands so far, still in progress.
T3’ T5’cSA ^Gal4-VP16 zp(A)
CryL BFP pA
S8.4A2 F1.1a ♀(+/-): IntegrationSequencing results (5’ only):
Integration in another vector line – B5.9a Gene: dynactin 1b
dynactin 1b is already hit in B5-9a, but not conditional.
Transcript 002 codes for 102 amino acids, corresponding to a.a. 18-119 in the mouse
ATGAGTTCAGATGGAGGTGGGC – dctn1bATG-F1
Transcript 001 is missing 114 amino acids
>chromosome:Zv9:13:11353643:11354423:1
ATATACTGCAGACGCAAACACTTCTCATGAACACAAACACACACACACACACACACACAC
ACACACACACACACACACACACACACACAGGTAAAGTGCTGTTTGTGTGATGCCATGCAA
CAGAGCGACTAAATACTGCCACTGCTATGGAAACAGAGGTTTGAGAAACATGCGGCACCT
TTTTGGGTTTGGTTCCTCGCTGCACACACAGATGGAGGAGGAAGAGGGACGGATTAGTGT dynactin1bS81-F1 Primer 1 (forward)TTGATCAGCCCGTGTGTTTGTAATGAGCATCAAAATCTCTCACATACAAACACATCAAGA
CTCACACCTAAACTGGACAGTCTTGGACAGAGGCCTTATAAAATGTCTGTGACAGCTTGG
GTTCATGCATGTTTGTGTTAGTGAGTGCATATTTGGGACCCCTTGTGAAAAGAAGTACAC
TAGATCATAGTTTTAAAAAGAGTACTTTTCATAGAAACGTTTCATATACTAAAGAGTTTA
CTCACCCTTCACTAGTATTAAACCTGTTGTGGGTTTGTTTCTTACATTGCACACAATTGC dynactin1bS8i-R1 Primer 2 (reverse) ATATTTGGAAGAATGTTGGAAACCGGTAACCATCCCAGCGAGCAAAATTCATGTGGCTTA
AATCAGGCCCACGCCAGACACTTACATCCGGCCCACATACTGCATAGAATGATGGCACTT
GGGTGGTCCACTCCTGTTTGCCAGAGCTAGGCCACAATTAAGCCATAGCAATACCACATA
TCAGCCAGAATTCAACCAAATGAACCAGAACTGACCCTATTCTGGGCCACAGTTTGCTTTT
S8.4A2 F1.1a ♀(+/-): PCR confirmation
Two PCRs performed (3rd PCR in progress): 3 primer Regular PCR – Forward/Reverse with Tol2F8 (5’) primer 2 primer Regular PCR – Forward primer (F1) & Tol2R3(3’) primer
Reverse primer (R1) & Tol2F8(5’) primer
3 primer PCR : 5 PCR tubes Gel Bands:
Labels:
GC = genomic control 1 (+/-) = vector (+/-) DNA with F1/R1 primers2 (+/-) = vector (+/-) DNA with F1/R1/Tol2F8(5’)
dynactin1bS8i-F1 Forward Primerdynactin1bS8i-R1 Reverse Primer
Results: * No mutant bands in vector (+) column (tube 4) * Gene trap integration not confirmed
2nd PCR (2 Primers)
S8.4A2 F1.1a ♀(+/-): PCR confirmation (contd)
2 primer PCR : 4 PCR tubes
Labels: Gel Bands:
1 (+/-) = vector (+/-) DNA with F1/Tol2R3(3’) primers2 (+/-) = vector (+/-) DNA with R1/Tol2F8(5’) primers
dynactin1bS8i-F1 Forward Primerdynactin1bS8i-R1 Reverse Primer
Results:* No mutant bands in vector (+) column (tube 3)* Gene trap integration is confirmed* No linkage to RFP expression
Still in progress: 3rd PCR with F1/R1/Tol2F10(5’) F1/R1/Tol2R4(3’)
Identified Loci : Integrations in Danio rerio
Fam100ab: Family with sequence similarity 100, member ab
- No biological evidence available in Danio rerio
Zmynd8: zinc-finger , MYND-type containing 8- Protein kinase C-binding protein 1 in humans- Considered a transcriptional regulator; studies done with mice and Xenopus – involved in neural differentiation (Zeng, 2009)
Dynactin 1b:- Dynactin is a multi-protein complex that enhances dynein activity (Zhapparova, 2009)- Phenotypes of human orthologue:
Neuropathy, distal hereditary motor, type VIIBPerry syndrome(Susceptible to) Amyotrophic lateral sclerosis
FUTURE GOALS More Inverse PCR
Work on new lines/vectors for zebrafish to identify loci – gene mappingB7.10A F1.1a (+/-) : In progress
1st vector : pcloa contamination Redo 3’ sequencing only – iPCR
Finish iPCR of 3’ sequencing for S8.4A2 F1.1a (+/-)
Continue 3rd PCR with 2 primers for S8.4A2 F1.1a (+/-)
ReferencesAsakawa, K., and Kawakami, K. (2009). The Tol2-mediated Gal4-UAS method for gene and enhancer
trapping in zebrafish. Methods, 49(3): 275-281.
Evans, M.J. (1998). Dev. Dyn., 212, 167-169 .
Hermanson, S., Davidson, A.E., Sivasubbu, S., Balciunas, D., and Ekker, S.C. (2004). Sleeping Beauty
transposon for efficient gene delivery. Methods Cell Bio. 77: 349-62.
Zeng, W., Kong, Q., Li, C., & Mao, B. (2010). Xenopus RCOR2 (REST corepressor 2) interacts with ZMYND8, which is involved in neural differentiation. Biochem Biophys Res Commun, 394(4): 1024-9.
Zhapparova, O. N., Bryantseva, S.A., Dergunova, L.V., Raevskaya, N.M., Burakov, A.V., Bantysh, O.B.,
Shanina, N.A., & Nadezhdina, E.S. (2009). Dynactin subunit p150Glued isoforms notable for differential interaction with microtubules. Traffic, 10(11): 1635-46.