In silico prediction of short hairpin RNA and in vitro silencing of activin receptor type IIB in chicken embryo fibroblasts by RNA interference

Vishnu, P. Guru; Bhattacharya, T. K.; Bhushan, Bharat; Kumar, Pushpendra; Chatterjee, R. N.; Paswan, Chandan; Dushyanth, K.; Divya, D.; Prasad, Athe Rajendra

doi:10.1007/s11033-019-04756-0

Cited by 7 publications

(11 citation statements)

References 35 publications

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“…We have not raised the positive birds to study gene knock-down by transferring shRNA constructs into the host genome. Earlier, transgenic knock-down birds were developed through other methods by transferring shRNA constructs for the speci c genes [33][34][35][36][37][38][39][40][41][42][43] . Thus, the protocol to develop shRNA based transgenic birds have been established through PGC mediated method.…”

Section: Discussionmentioning

confidence: 99%

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Production of Transgenic Chimeric Chicken from Cryopreserved Primordial Germ Cells and its Validation by Developing shRNA Transgenic Chicken Chimera

Divya

Shukla

Chatterjee

et al. 2021

Preprint

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Primordial germ cells (PGCs) are precursors of gametes in birds. For ex-situ conservation and production of transgenic birds, there is a limitation for preservation of oocytes in birds as compared to other mammalian species. To overcome those limitations, PGCs have been used as candidate cells, which have been cryopreserved and manipulated and used as to produce transgenic birds. In this study, cryo-preserved PGCs were used to produce transgenic birds. The protocol for production of transgenic birds with cryo-preserved PGCs was developed and the success rate for production of transgenics 16.7% in the protocols established in the study. The same gene transfer protocol through PGCs was validated by transferring shRNA molecule of SREBP-1 gene to the host genome to produce transgenic chimeric birds and the success rate for production of transgenic chimeric chicken was 40%. Finally, it is concluded that a standard protocol for ex-situ conservation of birds through PGCs and production of transgenic birds from cryo-preserved PGCs and knock down birds from PGCs were developed. It may be suggested that these protocols for resurrecting live birds from cryo-preserved PGCs may be applied as model for protecting the endangered birds from their extinction.

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Section: Discussionmentioning

confidence: 99%

“…AF047874) of size 119 bp by QGAPDHF: 5'-CTGCCGTCCTCTCTGGC-3' and QGAPDHR: 5'-GACAGTGCCCTTGAAGTGT-3'. The threshold cycle (Ct) value of each sample was noted, and average Ct values of each sample in duplicate qPCR reactions were estimated 37 .…”

Section: Gfp Expression Studymentioning

confidence: 99%

Production of Transgenic Chimeric Chicken from Cryopreserved Primordial Germ Cells and its Validation by Developing shRNA Transgenic Chicken Chimera

Divya

Shukla

Chatterjee

et al. 2021

Preprint

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“…However, the MSTN gene was silenced up to 68 and 75% in chicken embryo broblast cells (Sato et al 2006;Tripathi et al 2013). Later on, in the same chicken embryo broblast cells shRNA was used against MSTN, ACTRIIA, and ACTRIIB genes and observed the knockdown percentage of 68, 82 and 87, respectively (Tripathi et al 2013;Satheesh et al 2016;Guru Vishnu et al 2019). Even in in vivo studies, MSTN knockdown chicken showed 28% more body weight during 42 days of age compared to the control broiler chicken (Bhattacharya et al 2017).…”

Section: Acacb Gene Silencing In Chicken Myoblast Cellsmentioning

confidence: 99%

In vitro Silencing of Acetyl-CoA Carboxylase beta (ACACB) Gene Reduces Cholesterol Synthesis in Knockdown Chicken Myoblast Cells

Sushma¹,

Laxmi²,

Rao³

et al. 2021

Preprint

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The poultry industry provides cost-effective, healthy, and protein-enriched food for the growing population and achieving the nutritional security to the country. Excessive abdominal and subcutaneous fat deposition is one of the major setbacks to the poultry industry that reduces carcass yield and feed efficiency. In chicken abdominal fat constitutes 20% of total body fat which make up 2–3% of live weight of the bird. In fatty acid metabolism, acetyl-CoA Carboxylase (ACC) is one of the key enzymes with two isoforms i.e. ACACA and ACACB each of which plays a different role. In chicken, ACACB is involved in the β-oxidation of fatty acids and thereby potentially regulating the quality of meat and egg. The RNAi strategy is widely used for silencing the target gene expression. In this study, we designed five shRNA constructs and identified the most efficient shRNA molecule for silencing the ACACB gene under in vitro chicken embryo myoblast (CEM) primary cell culture system. After knocking down the ACACB gene, for understanding how fatty acid metabolism is regulated, we tracked the expression of key fatty acid metabolism genes like ACACA, FASN, SCD, ELOVL2, and CPT1. Also, checked the expression of immune response genes like IFNA, IFNB, and BLB1 in control as well as ACACB knockdown myoblast cells and observed no significant difference. We observed the down-regulation of key fatty acid metabolism genes along with ACACB, which may leads to the less fat accumulation in CEM cells. We also estimated the cholesterol and triglycerides in control and ACACB knockdown myoblast cells and found a significant difference between control and the knockdown cells. In vitro knockdown of the ACACB gene in a cell culture system by a short hairpin RNA (shRNA) expressing construct would help to produce a knockdown chicken with reduced fat deposition.

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“…Similarly, at the end of the RNAi experiment, six crabs from the RNAi and control groups were sampled, and their hepatopancreas and leg muscle tissues were collected for qRT-PCR detection of the target genes, including five ActRIIB signaling pathway genes (ActRI, SMAD3, SMAD4, forkhead box O (FoxO), and mammalian target of rapamycin (mTOR)) (Han et al, 2013;Guru et al, 2019), and three lipid metabolism-related genes (carnitine palmitoyltransferases 1β (CPT1 β), fatty acid synthase (FAS), and fatty acid elongation (FAE)) (Liu et al, 2016;DeBose-Boyd, 2018;Liu et al, 2018). The qRT-PCR procedures were similar to those for Es-ActRIIB expression detection.…”

Section: Expression Detection Of Target Genesmentioning

confidence: 99%

Molecular characteristic of activin receptor IIB and its functions in growth and nutrient regulation in Eriocheir sinensis

Wang

Zhang

Hou

et al. 2020

PeerJ

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Activin receptor IIB (ActRIIB) is a serine/threonine-kinase receptor binding with transforming growth factor-β (TGF-β) superfamily ligands to participate in the regulation of muscle mass in vertebrates. However, its structure and function in crustaceans remain unknown. In this study, the ActRIIB gene in Eriocheir sinensis (Es-ActRIIB) was cloned and obtained with a 1,683 bp open reading frame, which contains the characteristic domains of TGF-β type II receptor superfamily, encoding 560 amino acids. The mRNA expression of Es-ActRIIB was the highest in hepatopancreas and the lowest in muscle at each molting stage. After injection of Es-ActRIIB double-stranded RNA during one molting cycle, the RNA interference (RNAi) group showed higher weight gain rate, higher specific growth rate, and lower hepatopancreas index compared with the control group. Meanwhile, the RNAi group displayed a significantly increased content of hydrolytic amino acid in both hepatopancreas and muscle. The RNAi group also displayed slightly higher contents of saturated fatty acid and monounsaturated fatty acid but significantly decreased levels of polyunsaturated fatty acid compared with the control group. After RNAi on Es-ActRIIB, the mRNA expressions of five ActRIIB signaling pathway genes showed that ActRI and forkhead box O (FoxO) were downregulated in hepatopancreas and muscle, but no significant expression differences were found in small mother against decapentaplegic (SMAD) 3, SMAD4 and mammalian target of rapamycin. The mRNA expression s of three lipid metabolism-related genes (carnitine palmitoyltransferase 1β (CPT1β), fatty acid synthase, and fatty acid elongation) were significantly downregulated in both hepatopancreas and muscle with the exception of CPT1β in muscles. These results indicate that ActRIIB is a functionally conservative negative regulator in growth mass, and protein and lipid metabolism could be affected by inhibiting ActRIIB signaling in crustacean.

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In silico prediction of short hairpin RNA and in vitro silencing of activin receptor type IIB in chicken embryo fibroblasts by RNA interference

Cited by 7 publications

References 35 publications

Production of Transgenic Chimeric Chicken from Cryopreserved Primordial Germ Cells and its Validation by Developing shRNA Transgenic Chicken Chimera

Production of Transgenic Chimeric Chicken from Cryopreserved Primordial Germ Cells and its Validation by Developing shRNA Transgenic Chicken Chimera

In vitro Silencing of Acetyl-CoA Carboxylase beta (ACACB) Gene Reduces Cholesterol Synthesis in Knockdown Chicken Myoblast Cells

Molecular characteristic of activin receptor IIB and its functions in growth and nutrient regulation in Eriocheir sinensis

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