Cloning human SCN9/10A genes - Tips and Tricks?

I am looking to make some plasmid constructs with hNav1.7 and hNav1.8 in them. The company I’m working with has said that SCN genes from human are hard to clone. They cited this paper:

Voltage-gated sodium (Nav) channels control excitation in brain, heart, and muscle. Deviations
from their tightly defined functional characteristics, for example through genetic alterations, lead to
heritable pathology that reflects the spatial and temporal expression pattern of the affected Nav
channel gene ([1–7], reviewed in [13]). Pharmacological control of the clinical symptoms in these socalled Nav channelopathies (e.g., epilepsy, cardiac arrhythmia, muscle cramps, pain) is often hampered
by treatment-emergent adverse effects due to structural and functional similarities among the Nav
channels that make isoform-specific targeting difficult. Improved intervention may be possible with
patient-tailored therapy, where the drug action counters the defect of the faulty Nav isoform without
affecting other Nav channel function. Such drug development efforts are challenged by the unique
requirements that come with recombinant DNA work involving Nav channel coding sequences,
because Nav channels are notoriously difficult subjects when it comes to bacterial plasmid
amplification, mutagenesis, and stable cell line development. The basis for this behavior is unknown,
and no study researching the same has been published. One may speculate that the combination of
exceptionally large coding sequences with internal homologous repeats is involved. There is evidence
that Nav channel DNA in itself is toxic for the bacterial host, since random mutation events are not
limited to full-length Nav channel coding sequences, but they also occur during bacterial propagation
involving Nav channel DNA fragments (personal observation).

Does anyone have experience here? Obviously people achieve it. Here is at least one paper describing Nav1.7 plasmids constructs.
https://www.science.org/doi/10.1126/sciadv.aax4755

Any tips and/or experience?

@mamocol @tberta @thicunha @Nivedita

Hi Alex,
I know Ted Cummins has a human Nav1.7 in his lab. not sure about Nav1.8. I used genscript to synthesize the mouse nav1.7 sequence and clone it into a pcDNA3.1 plasmid. It’s pricey but it’s guaranteed to get you the constructs that you are looking for.

Thanks @AubinMoutal. Good to know. I’m going to go with the commercial gene synthesis approach, like you did for mouse. Worth it. I’ll let the vendor know that there has been success with mNav1.7

1 Like