Tol2 multisite gateway kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Tol2 MultiSite Gateway® kit is a molecular biology tool used for gene expression analysis and genetic manipulation. It facilitates the assembly of multiple DNA fragments into a single construct through a recombination-based cloning system. The kit provides the necessary components to enable efficient and streamlined creation of complex genetic constructs.

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Multisite Gateway (29 citations) Tol2 kit (5 citations) MultiSite Gateway Kit (4 citations)

2 protocols using tol2 multisite gateway kit

Tol2-Mediated DHX30 Variant Expression

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DHX30 cDNA plasmids were assembled using the Tol2 MultiSite Gateway® kit (Invitrogen, USA). Briefly, the cDNA of the wild-type DHX30 and DHX30 containing respective missense variants were amplified from the pEGFP-C3-DHX30 plasmids, using primers containing the appropriate att site sequences for BP recombination reactions. PCR products were purified and cloned into a pDONR221 donor vector using BP Clonase II enzyme mix following the manufacturer’s manual. The resulting middle entry clones pME-DHX30 were purified and verified by direct sequencing. To assemble the final expression plasmids, p5E-tuba1a promoter and pME-DHX30 were cloned into a Tol2-based destination vector, pDestTol2CG2 containing cmlc2:EGFP transgenesis marker, using LR Clonase II Plus enzyme mix following the manufacturer’s instructions. The resultant pTol2pA2-cmlc2:EGFP;tuba1a:DHX30 vectors were purified and verified by direct sequencing.

Mannucci I., Dang N.D., Huber H., Murry J.B., Abramson J., Althoff T., Banka S., Baynam G., Bearden D., Beleza-Meireles A., Benke P.J., Berland S., Bierhals T., Bilan F., Bindoff L.A., Braathen G.J., Busk Ø.L., Chenbhanich J., Denecke J., Escobar L.F., Estes C., Fleischer J., Groepper D., Haaxma C.A., Hempel M., Holler-Managan Y., Houge G., Jackson A., Kellogg L., Keren B., Kiraly-Borri C., Kraus C., Kubisch C., Le Guyader G., Ljungblad U.W., Brenman L.M., Martinez-Agosto J.A., Might M., Miller D.T., Minks K.Q., Moghaddam B., Nava C., Nelson S.F., Parant J.M., Prescott T., Rajabi F., Randrianaivo H., Reiter S.F., Schuurs-Hoeijmakers J., Shieh P.B., Slavotinek A., Smithson S., Stegmann A.P., Tomczak K., Tveten K., Wang J., Whitlock J.H., Zweier C., McWalter K., Juusola J., Quintero-Rivera F., Fischer U., Yeo N.C., Kreienkamp H.J, & Lessel D. (2021). Genotype–phenotype correlations and novel molecular insights into the DHX30-associated neurodevelopmental disorders. Genome Medicine, 13, 90.

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Tol2-mediated Transgenesis of DHX30

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DHX30 cDNA plasmids were assembled using the Tol2 MultiSite Gateway® kit (Invitrogen, USA).
Briefly, the cDNA of the wild-type DHX30 and DHX30 containing respective missense variants were amplified from the pEGFP-C3-DHX30 plasmids, using primers containing the appropriate att site sequences for BP recombination reactions. PCR products were purified and cloned into a pDONR221 donor vector using BP Clonase II enzyme mix following the manufacturer's manual. The resulting middle entry clones pME-DHX30 were purified and verified by direct sequencing. To assemble the final expression plasmids, p5E-tuba1a promoter and pME-DHX30 were cloned into a Tol2-based destination vector, pDestTol2CG2 containing cmlc2:egfp transgenesis marker, using LR Clonase II Plus enzyme mix following the manufacturer's instructions. The resultant pTol2pA2-cmlc2:EGFP;tuba1a:DHX30 vectors were purified and verified by direct sequencing.

Mannucci I., Dang N., Huber H., Murry J.B., Abramson J., Althoff T., Banka S., Baynam G., Bearden D., Beleza‐Meireles A., Benke P.J., Berland S., Bierhals T., Bilan F., Bindoff L.A., Braathen G.J., Busk Ø.L., Chenbhanich J., Denecke J., Escobar L., Estes C., Fleischer J., Groepper D., Haaxma C.A., Hempel M., Holler‐Managan Y., Houge G., Jackson A., Kellogg L., Keren B., Lemyre E., Kraus C., Kubisch C., Guyader G.L., Ljungblad U.W., Brenman L.M., Martínez-Agosto J.A., Might M., Miller D.T., Minks K.Q., Moghaddam B., Nava C., Chang T.C., Parant J.M., Prescott T., Rajabi F., Randrianaivo H., Reiter S.F., Schuurs-Hoeijmakers J., Shieh P.B., Slavotinek A., Smithson S., Stegmann A.P., Tomczak K.K., Tveten K., Wang J., Whitlock J.H., Zweier C., McWalter K., Friedman J.M., Quintero‐Rivera F., Fischer U., Yeo N.C., Kreienkamp H., & Lessel D. (2020). Genotype–phenotype correlations and novel molecular insights into the DHX30-associated neurodevelopmental disorders.

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