Plasmids containing the cDNA for human HIPKs were generously provided by two groups. Dr. Lienhard Schmitz gifted a plasmid containing hHIPK1 isoform 1, and Dr. Seong-Tae Kim provided us plasmids containing hHIPK3 isoform 2 and hHIPK4. The cDNA for hHIPK2 isoform 1 was synthesized by GenScript® to match the NCBI reference sequence NM_022740.4. In cases where the gifted cDNAs did not exactly correspond to the translated NCBI reference protein sequences (NP_938009.1 for hHIPK1, NP_001041665.1 for hHIPK3, and NP_653286.2 for hHIPK4), we performed site-directed mutagenesis using the GeneArt™ Site-Directed Mutagenesis PLUS system to correct the cDNA sequence. The cDNAs that corresponded to these reference sequences were then tagged with N-terminal Myc-epitope tags before being cloned into a pUAST-attB backbone vector using NotI and XhoI restriction sites for hHIPK1 and hHIPK2, BglII and KpnI sites for hHIPK3, and BglII and XhoI sites for hHIPK4. The four pUAST-attB-Myc-hHIPK plasmids were then sent to BestGene Inc. for injection into Drosophila embryos containing an attP40 site, allowing for stable integration to identical sites on the second chromosome. The resulting fly stocks each contain a single Myc-hHIPK cDNA under the control of a UAS promoter that is expressed in any cell expressing a Gal4 transcription factor.
Site directed mutagenesis plus system
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The Site-Directed Mutagenesis PLUS system is a laboratory equipment product that enables the introduction of specific mutations into DNA sequences. It provides a reliable and efficient method for generating targeted genetic modifications.
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2 protocols using site directed mutagenesis plus system
1
Generation of Drosophila HIPK Transgenic Lines
2
Generation of Myc-Tagged HIPK Fly Lines
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Plasmids containing the cDNA for human HIPKs were generously provided by two groups. Dr. Lienhard Schmitz gifted a plasmid containing hHIPK1 isoform 1, and Dr. Seong-Tae Kim provided us plasmids containing hHIPK3 isoform 2 and hHIPK4. The cDNA for hHIPK2 isoform 1 was synthesized by GenScript® to match the NCBI reference sequence NM_022740.4. In cases where the gifted cDNAs did not exactly correspond to the translated NCBI reference protein sequences (NP_938009.1 for hHIPK1, NP_001041665.1 for hHIPK3, and NP_653286.2 for hHIPK4), we performed site-directed mutagenesis using the GeneArt™ Site-Directed Mutagenesis PLUS system to correct the cDNA sequence. The cDNAs that corresponded to these reference sequences were then tagged with N-terminal Myc-epitope tags before being cloned into a pUAST-attB backbone vector using NotI and XhoI restriction sites for hHIPK1 and hHIPK2, BglII and KpnI sites for hHIPK3, and BglII and XhoI sites for hHIPK4.
The four pUAST-attB-Myc-hHIPK plasmids were then sent to BestGene Inc. for injection into D. melanogaster embryos containing an attP40 site, allowing for stable integration to identical sites on the second chromosome. The resulting fly stocks each contain a single Myc-hHIPK cDNA under the control of a UAS promoter that is expressed in any cell expressing a Gal4 transcription factor.
The four pUAST-attB-Myc-hHIPK plasmids were then sent to BestGene Inc. for injection into D. melanogaster embryos containing an attP40 site, allowing for stable integration to identical sites on the second chromosome. The resulting fly stocks each contain a single Myc-hHIPK cDNA under the control of a UAS promoter that is expressed in any cell expressing a Gal4 transcription factor.
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