Protocol detail

Cloning and expression of human GDF11 and FST

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The full coding sequence of human GDF11 was amplified by PCR using the following primers: 5’-CACCATGGTGCTCGCGGCCCCGCT-3’ and 5’-CGCTGTGGCTGCTCTTAA-3’. The product was then double-digested with NheI-HF and XbaI (New England Biolabs) and cloned into pcDNA3.1(+) (Life Technologies, Carlsbad, CA, USA). An alternate GDF11 expression construct was also made using the pRK5 vector (BD PharMingen) for experiments using HEK293T cells. Constructs for expression of wildtype human FST (isoform Fs288; in pcDNA3.1) (Cash et al., 2012a (link)) and human Furin (in pcDNA4; Walker et al., 2017 (link)) were previously described. Site-directed mutagenesis was employed to generate the respective expression vectors carrying the GDF11:c.893G>A and FST:c.167G>A variants. Separately, four SMAD-binding elements (SBE, 5’-CAGACA-3’) were synthesized (IDT, Coralville, IA, USA) and cloned into pENTR/D-TOPO and then shuttled into a cFos-FFLuc plasmid (SBE-FFLuc) for the in vitro luminescence reporter activity assay.

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Cox T.C., Lidral A.C., McCoy J.C., Liu H., Cox L.L., Zhu Y., Anderson R.D., Uribe L.M., Anand D., Deng M., Richter C.T., Nidey N.L., Standley J.M., Blue E.E., Chong J.X., Smith J.D., Kirk E.P., Venselaar H., Krahn K.N., van Bokhoven H., Zhou H., Cornell R.A., Glass I.A., Bamshad M.J., Nickerson D.A., Murray J.C., Lachke S.A., Thompson T.B., Buckley M.F, & Roscioli T. (2019). Mutations in GDF11 and the extracellular antagonist, Follistatin, as a likely cause of Mendelian forms of orofacial clefting in humans. Human mutation, 40(10), 1813-1825.

Publication 2019

NheI-HF XbaI pcDNA3.1(+) pRK5 vector

Cells Coding sequence Furin Gdf11 Human Isoform Luminescence assay Plasmid Primers Site directed mutagenesis Topo Vectors Walker

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Variable analysis

independent variables

GDF11 expression construct (pcDNA3.1(+) or pRK5 vector)
GDF11:c.893G>A variant
FST:c.167G>A variant

dependent variables

Luminescence reporter activity (SBE-FFLuc)

control variables

Wildtype human GDF11 expression construct
Wildtype human FST (isoform Fs288) expression construct
Human Furin expression construct

controls

Positive control: Wildtype human GDF11, FST, and Furin expression constructs
Negative control: Not explicitly mentioned

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