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Fly Genetics: Exploring MEK Variants

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Fly stocks were maintained under standard conditions and crosses were performed at 25°C unless otherwise specified. OregonR, Histone-GFP, MTD-GAL4, UAS-MEK^WT, UAS-MEK^F53S, UAS-MEK^Y130C, and UAS-MEK^E203K flies were described in Jindal et al. (2017b) (link). S218A/S222A were introduced into UAS-MEK^F53S construct to generate UAS-MEK^{F53S SSAA} with site-directed mutagenesis using Q5 hot start DNA Polymerase (NEB M0493) with the primers mek_218_228S2A_F: CCAACgCCTTTGTGGGCACCCGTA and mek_218_228S2A_R: CCATCGcGTCGATCAGTTGACCGGAG and verified by sequencing. The UAS-MEK^{F53S SSAA} construct was integrated into attP40 site using the ΦC31-based integration system. UAS-ksr RNAi (41598) and UAS-RAF RNAi (41589) were from the Bloomington Drosophila Stock Center. The UAS-Mkp3 RNAi line was generated by integrating a previously established RNAi construct HMS04475 (a gift from Norbert Perrimon) onto the third chromosome by ΦC31 integration into attP2 (Sopko et al., 2014 (link)). The newly developed fly lines are available on request. Egg lays and embryonic development were conducted at 22°C.

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Marmion R.A., Yang L., Goyal Y., Jindal G.A., Wetzel J.L., Singh M., Schüpbach T, & Shvartsman S.Y. (2021). Molecular mechanisms underlying cellular effects of human MEK1 mutations. Molecular Biology of the Cell, 32(9), 974-983.

Publication 2021

Q5 hot start DNA Polymerase

Chromosome Dna polymerase Drosophila Embryonic development Histone Primers Rnai Site directed mutagenesis

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Variable analysis

independent variables

MEK genotypes (WT, F53S, Y130C, E203K, F53S SSAA)

dependent variables

Not explicitly mentioned

control variables

Standard conditions for fly stock maintenance
Temperature at 25°C unless otherwise specified
Egg lays and embryonic development conducted at 22°C

controls

Positive control: OregonR fly line
Negative controls: UAS-ksr RNAi, UAS-RAF RNAi, UAS-Mkp3 RNAi

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