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.