In silico prediction of short hairpin RNA and in vitro silencing of activin receptor type IIB in chicken embryo fibroblasts by RNA interference
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Gene silencing by RNA interference is extensively used reverse genetic approach to analyse the implications of any gene in mammalian systems. The silencing of the Activin type IIB receptor belonging to transforming growth factor beta superfamily has demonstrated increase in muscle growth in many species. We designed five short hairpin RNA constructs targeting coding region of chicken ACTRIIB. All the shRNAs were transfected into chicken embryo fibroblast cells and evaluated their silencing efficiency by real time PCR and western blotting. Initially the computational analysis of target region and shRNA constructs was undertaken to predict sequence based features (secondary structures, GC% and H-b index) and thermodynamic features (ΔGoverall, ΔGduplex, ΔGbreak−target, ΔGintra−oligomer, ΔGinter−oligomer and ΔΔGends). We determined that all these predicted features were associated with shRNA efficacy. The invitro analysis of shRNA constructs exhibited significant (P < 0.05) reduction in the levels of ACTRIIB at mRNA and protein level. The knock down efficiency of shRNAs varied significantly (P < 0.001) from 83% (shRNA 1) to 43% (shRNA 5). All the shRNAs up regulated the myogenic pathway associated genes (MyoD and MyoG) significantly (P < 0.05). There was significant (P < 0.05) up-regulation of IFNA, IFNB and MHCII transcripts. The ACTRIIB expression was inversely associated with the expression of myogenic pathway and immune response genes. The anti ACTRIIB shRNA construct 1 and 3 exhibited maximum knock down efficiency with minimal interferon response, and can be used for generating ACTRIIB knockdown chicken with higher muscle mass.
KeywordsMuscle growth ACTRIIB Secondary structures Thermodynamic parameters Gene expression Gene silencing
All the authors would like to thank to the Indian Council of Agricultural Research (ICAR) for giving financial assistance in carrying out this experiment under National Fellow project.
P. Guru Vishnu conducted wet lab experiment. T.K. Bhattacharya guided the whole experiment, designed oligos and prepared the manuscript. P. Kumar and Bharat Bhushan assisted to analyse data. R.N. Chatterjee and Chandan paswan prepared the tables and figures. K.Dushyanth, D.Divya and A.Rajendraprasad helped in compiling the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
We declare that the whole experiment conducted was conforming to the ethical standards and was approved by Institutional Animal Ethics Committee (IAEC) of ICAR-Directorate of Poultry Research, Hyderabad.
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