Isolation Of Goat Skeletal Satellite Cells, Cloning Expression And Functional Exploration Of MyoD1and MyoG

It was widely acepptted that Myogenic regulatory factors (MRFs), which are basic helix-loop-helix (bHLH) transcription factors, play the irreplaceable roll in the myogensis of skeletal. The function of MyoDl and MyoG were not only guarantee of skeletal development, but also the economic guarantee of the meat animals. The MRFs could be divided into two type. The first type was determination-class MRFs, which was consist of MyoDl and Myf5and in charge of the determination of skeletal pregenitor cell during the early embryonic period. The other group was differentiation-class MRFs, which consist of MyoG and Myf4. The biofunction research of MRFs were raletive clear in mouse and human being, but few biofunction researches were carried out on the sheep or goat. In this paper, the complete CDS of MyoDl and MyoG were cloned and ectopic expressed in goat dermal fibroblast to explore the biofunction of MyoD1by reconstruction of the eukaryotic expression vector. At last, MyoD1transgenic sheep and MyoG transgenic goat were produced to obtain new species for animal breeding by testicular injection. Major contents in this study are as follows:1. Isolation and Myo-differention of Goat Skeletal Satellite Cells Goat skeletal satellite cell (gSMSCs) of high purity were isolated by improving the isolation method according to their specific cell location which was between the basement-membrane and serosa. The muscle fiber were firstly obtained under stereo microscope. Then, cell in the muscle fiber were released by breaking down of the fiber. At last, skeletal satellte cells were purified by the6-succession passage with different adherence time for the high proliferation rate and long adherence time. The purity were over90%(Pax-7+rate was92.5±3.4%; MyoD+rate was93.0±1.8%), which identified via IFA. The myo-differentiation of gSMSCs were carried out with two differention media. The myo-differention results suggest Opti-MEM were rather fitter for myo-differention of gSMSCs. The IFA resuls of sSMSCs indicated92%were in the differentiation procedure for the Desmin+(92.3±1.1%) and MyHC+(93.0±1.2%) rate.2. cloning and bioinformatics analysis of MyoDl and MyoG Total RNA of gSMSCs on myo-differetion strating period were extracted on by TriZol reagent. After the inverse transcription of total RNA, the complete CDS of MyoDl and MyoG were cloned by PCR with help of PrimeSTAR HS DNA polymerase. The CDS were sequenced by Invitrogen (shanghai) after T-A cloning. Then the result were submitted to NCBI-BLAST and the results showed that MyoDl and MyoG were in rather high homology among species, what’s more, it was the same that the animo acid sequence of MyoDl (or MyoG) between sheep and goat. The tools online analysis results indicated that MyoDl and MyoG were nucleus located and hydrophobic protein without any signal peptide. The NCBI-CCD analysis results remind that MyoD1and MyoG contain a bHLH structure, while MyoDl also had a Myf5specific structure.3. Construction and Myo-differentiation of MyoDl Ectopic Expression Goat Dermal Fibroblast line goat skin fibroblast (GSF) were isolated by tissue inoculate, and pEGFP-MyoD1eukaryotic expression vector were constructed by molecular biology methods. MyoD1ectopic expression GSF line were established by transfected of pEGFP-MyoD1eukaryotic expression vector via LipofectaminTX LTX and by14-day400μg/mL G418succession screen. At last, MyoDl ectopic expression GSF were identified by IFA. The myo-differentiation of MyoDl ectopic expression GSF were analyzed by detecting the expression of Desmin and MyHC via IFA. The results showed that90%cell went into the myo-differention procdure.4. Construction of MyoG eukaryotic expression vector and production of anti-MyoG serum via genetic immunization MyoG eukaryotic expression vector (pcDNA3.0-MyoQ pEGFP-MyoG) were constructed via molecular biology methods. After coated by polypropyleneimine (PEI) at1μg:1.5μg,100μg pcDNA3.0-MyoG were injected into each mouse quadriceps femoris4time every2week, to produce anti-MyoG serum. The IFA and Western Blot results indicated that the anti-serum could specificly recognize the MyoG protein and be used in the later experiment 5. Production of MyoD1Transgenic Sheep and MyoG Transgenic Goat After being lineared by MIu I, Plasmids (pEGFP-MyoDl or pEGFP-MyoG) were coated by PEI at1:2to obtain the plasmid-PEI complex.300μg complex were injected into the mouse mice twice in one week.10day after the sencond injection, the males were mate with female. After finishing mating, mice were finished to obtain the testis and spermatozoon. It was founded that there were some green substance in the ductus epididymis and the spermatozoon head were filled of green substance. As the mice, testicular injection were carried out to obtain tansgenic sheep or goat.3mg complex were injected into each side of sheep (or goat) testis. A sceond injection were carried out1week later.40dyas later, the treated sheep or goat were mate with females. Finally,4MyoDl-eGFP transgenic sheep and13eGFP-MyoG transgenic goats of the first generation were obtained, with positive rates of6.7%(4/59) and21.67%(13/60) respectively.