Generating Mutant Kdm5b and Kdm4d Constructs

The cDNA encoding wild-type Kdm5b was obtained from Addgene (86398)20 (link). To generate a catalytically inactive version of Kdm5b the H499A mutation was introduced by site directed mutagenesis. The in vitro transcription plasmids containing the wild-type and mutant Kdm4d were obtained from Addgene (61553, 61554)25 (link). mRNA was in vitro transcribed with T7 or T3 mMESSAGE mMACHINE kits (Ambion) and purified by LiCl precipitation. All plasmids generated in this study are available at Addgene under “Torres-Padilla lab plasmids”.

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Borsos M., Perricone S.M., Schauer T., Pontabry J., de Luca K.L., de Vries S.S., Ruiz-Morales E.R., Torres-Padilla M.E, & Kind J. (2019). Genome-lamina interactions are established de novo in the early mouse embryo. Nature, 569(7758), 729-733.

Publication 2019

T7 or T3 mMESSAGE mMACHINE kits

Cdna Kdm5b Mrna Mutation Plasmids Site directed mutagenesis Transcription

Corresponding Organization :

Other organizations : Helmholtz Zentrum München, University Medical Center Utrecht, Ludwig-Maximilians-Universität München

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Variable analysis

independent variables

H499A mutation to generate catalytically inactive version of Kdm5b

dependent variables

Not explicitly mentioned

control variables

Wild-type Kdm5b
Wild-type and mutant Kdm4d

controls

Positive control: Wild-type Kdm5b
Negative control: Catalytically inactive mutant Kdm5b (H499A)

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