生物工程外文翻譯--rnai沉默肌肉生長抑制素基因致斑馬魚表型巨大化（英文）

1、Journal of Biotechnology 119 (2005) 324–331Myostatin gene silenced by RNAi show a zebrafish giant phenotypeJannel Acosta 1, Yamila Carpio 1, Ingrid Borroto, Osmany Gonz´ alez, Mario Pablo Estrada ?Aquatics Biotechno

2、logy Project, Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology, P.O. Box. 6162, Habana 10600, CubaReceived 23 December 2004; received in revised form 15 April 2005; accepted 27 April 2005Ab

3、stractMyostatin is a member of the transforming growth factor-? (TGF-?) family that functions as a negative regulator of skeletal muscle development and growth. Recently, it has been reported that the transgenic zebrafis

4、h expressing myostatin prodomain exhibited an increased number of fiber in skeletal muscle. Other novel results suggest that myostatin plays a mayor role during myogenesis, apart from inhibition of proliferation as well

5、as differentiation. We have investigated the ability of double-stranded RNA (dsRNA) to inhibit myostatin function in the zebrafish. By microinjection dsRNA, corresponding to biologically active C-terminal domain from ami

6、noacid 268 to end codon of tilapia myostatin protein, we produced an increased body mass in treated fish. The dsRNA injection in early development stage in zebrafish produced hyperplasia or hypertrophy. In addition, the

7、interference of gene function showed a strong dependence on the amount of dsRNA. © 2005 Elsevier B.V. All rights reserved.Keywords: Zebrafish; Myostatin; RNAi; Gene silencing; Hypertrophy; Hyperplasia1. Introduction

8、The myostatin (MSTN) is a member of the trans- forming growth factor-? (TGF-?) and it has a pivotal role as a negative regulator of skeletal muscle growth in several mammalian species.? Corresponding author. Tel.: +53 7

9、2718466x5154; fax: +53 7 2731779. E-mail address: mario.pablo@cigb.edu.cu (M.P. Estrada). 1 Jannel Acosta and Yamila Carpio contributed equally to this work.The “Double Muscle” breeds of cattle that have significantly mo

10、re muscle mass than standard breeds were found to carry natural mutations in the myo- statin gene (McPherron and Lee, 1997; Kambadur et al., 1997; Grobet et al., 1997, 1998). Myostatin knock- out mice show a dramatic inc

11、rease of skeletal muscle mass, and the increase results from a combination of hyperplasia and hypertrophy (McPherron et al., 1997). In 2001, Lee and McPherron reported that transgenic mice expressing the myostatin prodom

12、ain, follistatin or dominant-negative form of activin receptor type IIB0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.04.023326 J. Acosta et al. / Journal of Biot

13、echnology 119 (2005) 324–331cDNA was synthesized in 20 ?l reverse transcription (RT) reaction from 5 ?g of total RNA by using reverse transcription system (Promega). PCR was performed with 20 ?L of the 1:5 dilution of RT

14、 reaction. The primers used to PCR amplify the 496 bp of zebrafish myostatin and Max 476 bp cDNA (used as an internal standard to normalize differences in template amounts) were the same used by Amali et al. (2004). The

15、PCR program was: an initial denaturation at 94 ?C for 2 min, a 35 cycles for myostatin and 30 cycles for Max was performed at 94 ?C for 30 s, 55 ?C for 30 s and 72 ?C for 1 min. The final extension time was 7 min at 72 ?

16、C. The PCR products were subjected to 1% gel elec- trophoresis. The intensities of the bands were quan- tified on 1D Image Analysis Software (Kodak) and used for the calculation of the relative gene expression values. Th

17、e cycle number for the gene expression study was determined by a validation test in which the PCR was performedasdescribedbutterminatedatdifferentcycle number. A kinetic profile of the amount of PCR product generated at

18、different PCR cycles was constructed and the cycle number used was chosen within the exponen- tial region of the amplification curve. This is to ensure that the amount of the PCR product reflects the amount of template i

19、n the original sample.2.3. Growth evaluation and muscle histological analysisTo determine the body weights all fish were weighted at 30, 60 and 75 days post-microinjection. For muscle histological analysis, twenty indivi

20、dual fish from each group were fixed in formol for 10 h followed by routine paraffin sectioning and Haematoxilin/Eosin staining. A vertical section at the base of the first pin of the pelvic fin was selected for quantifi

21、cation of the number of muscle fibers. Stained sections were pho- tographed and the images were analyzed using DIGI- PAT program (version 3.3, Eicisoft, Cuba). The total number of fibers in a given area was determined by

22、 counting them under a microscope at a fixed magnifi- cation (20×).2.4. Statistical analysisError bars indicate standard error of the mean. (**) indicates that P < 0.01 and (*) P < 0.05, for Student’st-test co

23、mparing the microinjected group to control group.3. Results and discussionRecently the tilapia (Oreochromis mossambicus) myostatin gene (GenBank accession no. AF197193) was identified and cloned. The nucleic and amino ac

24、id sequences from tilapia are highly homologous to zebrafish, which is approximately 74% identical to tilapia DNA sequence (Rodger et al., 2001). We generated dsRNA corresponding to the biologi- cally active C-terminal d

25、omain from the aminoacid 268 to end codon of the tilapia myostatin protein. To determine whether tilapia myostatin dsRNA is capable of stimulate zebrafish growth, the dsRNA syn- thesized was introduced into zebrafish emb

26、ryos using standard microinjection techniques and the embryos grown for assay. Exposure of a parental animal to only a few molecules of dsRNA per cell triggered gene silenc- ing throughout the treated animal (Hannon, 200

27、2). We assayed two doses of dsRNA, 5 and 5 × 106 molecules of dsRNA per embryo. In order to determine the body weight gain, we weighted all fish at 30, 60 and 75 days post- microinjection and compared both treated g

28、roups with their controls. At 75 days post-microinjection the aver- age body weight of microinjected fish with 5.5 and 5.5 × 106 molecules of dsRNA was increased by 39 and 45%, respectively, compared with their cont

29、rols (Fig. 1; Table 1). The phenotype observed was con- sistent with transgenic zebrafish expressing myostatin prodomain, but the increase of body weight was higher than homozygous transgenic zebrafish, which have 10–15%

30、 of increase in body mass compared with non- transgenic fish (Xu et al., 2003). In concordance with previous results (Li et al., 2000), we observed a dose-response for generation of the phenotypes expected. The animal mi

31、croinjected with 5 × 106 molecules of dsRNA showed a more dramatic increase in average body weight than those injected with five molecules. Double-stranded RNA injection did not produce obvious nonspecific defects,