RT‐PCR‐Based Cloning of Human SMN, the SMA Determining Gene, and the Construction of Its Expression Plasmids

Abstract

Spinal muscular atrophy (SMA) is a lethal autosomal recessive disease. The gene most highly associated with SMA is the survival motor neuron (SMN) gene. To study the biological function of SMN, the availability of full‐length SMN protein is essential. For that reason, the purpose of the study was to develop a new procedure for the cloning of human SMN, the SMA‐determining gene. Unlike the pre‐existing cloning method, the new approach is based on the reverse transcription (RT) and the polymerase chain reaction (PCR); it is also cost‐effective, not time‐consuming, and suited for any laboratory interested in SMA research.

The development of this RT‐PCR–based cloning procedure may lead to a new procedure for the construction of expression plasmids, using the pFastBac™ HTb and the pBlueBacHis2 A transfer vectors for the purpose of obtaining human SMN protein in insect cells, and using the pET‐28a (+) transfer vector for the purpose of obtaining human SMN protein in bacteria.

The results of this research offer an easier way to obtain full‐length recombinant SMN protein, which is valuable for biochemical and biological analyzes that may elucidate the molecular mechanism of SMA. Knowing the molecular mechanism of SMA will allow the exploration of gene therapy in SMA.

Keywords:

• RT‐PCR reactions
• cloning
• spinal muscular atrophy
• survival motor neuron gene
• expression plasmids