通过RNA干扰降低肌肉生长抑制素基因,增加鸡体重

1、1Introduction1.Growth is regulated by a number of genes of which mostly allthe genes except myostatin controls growth by increasing musclemass in chicken.2.The lack of myo-statin function results in the excessive growth of skeletal muscle,which delineates the existence of a powerful mechanism to con

2、trolmuscle size in animals3.During embryonic stage,myostatin down regulates Pax-3 gene,which is associated withproliferation of myogenic cells.24.Increased expression ofmyostatin was also observed in humans with chronic illness, HIVinfection and early aging due to muscle atrophy.5.The limitingof fun

3、ction of negative regulator of any trait can be achieved by anumber of techniques of which shRNA based knock down approachwould be a promising one. 6.In this study, chicken has been used asmodel organism to estimate the effect of shRNA on phenotypes.3AnimalsControl broiler birds maintained at the In

4、stitute farmSlaughter of birds was performed fol-lowing standard protocol of cervical dislocationMSTN gene (Accession No. AF346599)Fertile eggs of control broiler chicken line werecollected from the Institute farm,scrambled and control birds using Trizol accord-ing to manufacturers instruction (Sigm

5、a).The analysis kit (Jeev Diagnos-tics Pvt. Ltd, India)Experimental Materials4Experiment processDesigning and cloning of shRNA oligosTransfection of embryo by manipulationSperm mediated method of transfer of shRNA moleculesScreening of positive knock-down birds by PCR and southernblottingGrowth, car

6、cass and biochemical traitsStatistical analysis5Fig. 1. Secondary structures of antisense strands of shRNAs for myostatin gene predicted at the site6Fig. 2. Location of ink in the yolk inoculated through 26 gauge needle from narrow end of egg. A length of 2 cm of the needle inserted into the egg and

7、 spot shows the locationof ink in the yolk of the egg. 7The 4 day The 5 dayThe 6 dayThe 7 dayControl groupFertility 8482807786Fattening 76.171.972.568.891.8 Hatchability 6459585379%The hatchability percentage for devel-opment of knock down chicks was 58%. Results8DMSOElectroporation methodElectropor

8、ation with PBS bufferlipofection methodControl groupFertility 54824664 70Fattening 81.4 92.6 47.837.576.2 Hatchability 44%76%22%24%53.3%the fertility for developing knock down chicks from spermmediated method was 41.5%.9Fig. 3. PCR product in screening of positive knock down birds amplified from DES

9、Tvector.10Fig. 4. Southern blot of genomic DNA digested with PstI enzymecollected fromknock down chicken containing shRNA molecules Mol1 to Mol5 including scrambledand control group.11Fig. 5. The mRNA expression level in knock down birds with 5 shRNA molecules, birds of scrambled shRNA molecule and

10、control group revealed differential expressionamong groups where birds of molecule4 had the lowest expression of MSTN mRNA12Fig. 6. Western blot of myostatin expressed in breast muscle tissue collected from knock down chicken containing shRNA molecules Mol1 to Mol5 including scrambled andcontrol gro

11、up.13Fig. 7. In-situ histochemistry of breast muscle of knock down chicken carrying shRNA 4 molecule revealing blot of MSTN protein inside the myoblast cells. 1415Discussion1.The introduction of shRNA on day 4 of embryonic stageprovided the best hatching performance to produce chick and theday progr

12、essed, the hatching rate was reduced gradually.2.The efficiency of different shRNA molecules forknocking down the expression of MSTN protein revealed relativelybetter activity by the molecule 3 followed by molecule4, molecule5,molecule2 and molecule1.3.Electroporation with HT or PBS and DMSOsimple i

13、ncubation may be used for better transfection and ultimateeffect 164.lower mRNA expression of myostatin gene inbreast muscle of knock-down chicken than that of control as wellas scrambled birds5.In this experiment, body weights at different ages upto 6 weekswere higher in knock-down birds as compared to control birds,in which molecule2 performed the best.6.Thus, our approach of use of shRNA against myostatin gene hasenhanced body weight of birds at juvenile age.17AcknowledgementsThe work was conducted und