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Donor and helper plasmids for transgenesis in vertebrates.

Tol1-based transgenesis vector

Background

The donor plasmid contains terminal regions of the Tol1 element and multicloning sites for integration of a gene to be transferred to the host chromosome. The helper plasmid carries the transposase gene of the Tol1 element. Tol1 is a DNA transposon identified in the medaka fish and demonstrated to be active in various vertebrate species.

Kit component

pDon122: A vacant donor plasmid.

pDon123: Donor plasmid carrying the GFP gene.

pHel105: Helper plasmid. Its vector portion is pCS2+, having the CMV promoter for in vivo expression of the transposase gene and the SP6 promoter for in vitrosynthesis of the transposase mRNA.

pHel106: A defective helper which is useful for negative control experiments especially when you want to know the net transformation efficiency.


a.Target site duplication
CCTTTAGC

b. Tol1 left arm
nt. 1-157 of GenBank D42062

c. Multicloning sites
GATCC GAATTC GATATC GGTACC CTGCAG TCTAG
BamHI EcoRI EcoRV KpnI PstI XbaI

d. Tol1 right arm
nt. 1750-1855 of GenBank D42062

e. Tartget site duplication
CCTTTAGC

f. pUC19
GenBank U55763.
nt. 391-460 were changed to:
CCAGTGAAT GTCGAC CATGC AAGCTT GGCGTAAT
HincII HindIII

g. CMV promoter + EGFP + Poly(A) signal
nt. 1-1632 of GenBank U55763
Inserted at the EcoRV site of the muticloning sites.

h. Entire coding sequence for transposase
nt. 31-2817 of GenBank AB264112

i. pCS2 (+), carrying CMV promoter, SP6 promoter, MCS 1, 3' UTR, and MCS2.

j. nt. 31-995 of GenBank AB264112

k. nt. 996-1001 (ATGAAA for methionine and lysine) were changed to two stop codons (TAGTAA).

l. nt. 1002-2817 of GenBank AB26411

<How to recover plasmid DNA>

1. Cut out one of the circles of the paper and immerse it in water or TE in a microfuge tube. Other circles are for backup.

2. Mix by tapping.

3. Centrifuge for 1 minute at >10 krpm.

4. Transform competent bacterial cells (commonly used strains, such as JM109, DH5a and XL1-Blue) with a small amount of supernatant.

5. Spread the bacteria on an LB/agar plate containingl ampicillin, and incubate the plate at 37 oC for >12 hours.

6. Pick up a single colony.

7. Amplify bacteria in liquid media.

8. Extract plsmid DNA by the standard method.

<Reference>
1) Koga A, Cheah FS, Hamaguchi S, Yeo GH, Chong SS (2008). Germline transgenesis of zebrafish using the medaka Tol1 transposon system. Dev Dyn. 237: 2466-2474.
2) SKoga A, Higashide I, Hori H, Wakamatsu Y, Kyono-Hamaguchi Y, Hamaguchi S (2007). The Tol1 element of medaka fish is transposed with only terminal regions and can deliver large DNA fragments into the chromosomes. J. Hum. Genet. 52: 1026-1030.

References

1) Koga A, Cheah FS, Hamaguchi S, Yeo GH, Chong SS (2008). Germline transgenesis of zebrafish using the medaka Tol1 transposon system. Dev Dyn. 237: 2466-2474.
2) SKoga A, Higashide I, Hori H, Wakamatsu Y, Kyono-Hamaguchi Y, Hamaguchi S (2007). The Tol1 element of medaka fish is transposed with only terminal regions and can deliver large DNA fragments into the chromosomes. J. Hum. Genet. 52: 1026-1030.

Product List

Product Name Cat# Quantity Price

Tol1-based transgenesis vector

CSR-CT-NU-002-1 1TEST

¥ 50,000
$ 667
€ 500

1) Koga A, Cheah FS, Hamaguchi S, Yeo GH, Chong SS (2008). Germline transgenesis of zebrafish using the medaka Tol1 transposon system. Dev Dyn. 237: 2466-2474. 2) SKoga A, Higashide I, Hori H, Wakamatsu Y, Kyono-Hamaguchi Y, Hamaguchi S (2007). The Tol1 e