Plasmid vectors with either RSV or CMV promoter are frequently used for DNA- mediated immunization due to the availability in commercial. Consequently, influence of the vector constituents, such as promoter, enhancer and transcription termination signal etc. on vaccination efficiency is not studied extensively. As an initial attempt to develop an efficient vector system for DNA-rnediated immunization, influence of promoter for antigen gene expression on vaccination efficiency has been analyzed. Initially, plasmids with either B-actin or muscle creatine kinase (MCK) promoter were constructed from the plasmid with prototype CMV promoter. In addition, ovalbumin (OVA) antigen gene has been cloned into each vectors to generate the plasmid vectors with different promoters for induction of the anti-OVA immune responses. Antigen protein expression in antigen gene transfected mouse muscle myoblast cells showed that the level from MCK promoter containing plasmid was slightly higher than those from either CMV or B-actin promoter containing plasmids. Also, the same plasmid turned out to be slightly more efficient than other plasmids in antibody imrnune response induction in vivo, when they were applied both through intramuscularly and intradermally. These results suggest that the commonly used CMV promoter containing plasmid vector could be further modified to develop an efficient vector for DNA-mediated immunization.
Animals ; Clone Cells ; Creatine Kinase, MM Form ; Gene Expression ; Immunization ; Mice ; Myoblasts ; Ovalbumin ; Plasmids* ; Vaccination*

OBJECTIVEDiscusses the distributive characters of the Creatine Kinase MM (CKMM) gene A/G Polymorphism in XinjiangUyghur, One hundred and fourtheen athletes and 441 general population of Uyghur were involved in the study.

METHODSPolymerase chain reaction-restriction fragment length polymorphism was used.

RESULTS(1) The CKMM gene A/G frequency in Uyghur general population was(AA, AG and GG) 0.497, 0.392 and 0.111, the result test by Hardy-Weinberg (H-W) equilibrium and x² = 2.72, P = 0.1, df = 2, indicated that the control group had representative. (2) AA, AG and GG genotype frequency of power-oriented athlete respectively was 0.442,0.302 and 0.256, frequency of GG genotype and G allele was higher than the control group, there were significant differences compared to thecontrol( P < 0.05, df = 2); (3) A/G genotype frequency of Endurance-oriented athletere spectively was 0.571, 0.400 and 0.029, there were nosignificant differences compared to the controls ( P > 0. 05, df = 2). (4) A/G genotype frequency of Uyghur soccer athletes respectively was0.472, 0.361 and 0.167, G allele was higher than the Endurance-oriented athlete and lower than the power-oriented athletes. and no significant differences compared to the controls( P > 0.05, df = 2).

CONCLUSIONThe results indicate that the CKMM gene GG genotype and G alleleare represented in power-oriented athletes, but don't find A/G polymorphism correlation with endurance and the football sport performance.

Alleles ; Asian Continental Ancestry Group ; genetics ; Athletes ; Athletic Performance ; China ; Creatine Kinase, MM Form ; genetics ; Gene Frequency ; Genotype ; Humans ; Physical Endurance ; genetics ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length

Sumoylation, the conjugation of a small ubiquitin-like modifier (SUMO) protein to a target, has diverse cellular effects. However, the functional roles of the SUMO modification during myogenesis have not been fully elucidated. Here, we report that basal sumoylation of histone deacetylase 1 (HDAC1) enhances the deacetylation of MyoD in undifferentiated myoblasts, whereas further sumoylation of HDAC1 contributes to switching its binding partners from MyoD to Rb to induce myocyte differentiation. Differentiation in C2C12 skeletal myoblasts induced new immunoblot bands above HDAC1 that were gradually enhanced during differentiation. Using SUMO inhibitors and sumoylation assays, we showed that the upper band was caused by sumoylation of HDAC1 during differentiation. Basal deacetylase activity was not altered in the SUMO modification-resistant mutant HDAC1 K444/476R (HDAC1 2R). Either differentiation or transfection of SUMO1 increased HDAC1 activity that was attenuated in HDAC1 2R. Furthermore, HDAC1 2R failed to deacetylate MyoD. Binding of HDAC1 to MyoD was attenuated by K444/476R. Binding of HDAC1 to MyoD was gradually reduced after 2 days of differentiation. Transfection of SUMO1 induced dissociation of HDAC1 from MyoD but potentiated its binding to Rb. SUMO1 transfection further attenuated HDAC1-induced inhibition of muscle creatine kinase luciferase activity that was reversed in HDAC1 2R. HDAC1 2R failed to inhibit myogenesis and muscle gene expression. In conclusion, HDAC1 sumoylation plays a dual role in MyoD signaling: enhancement of HDAC1 deacetylation of MyoD in the basally sumoylated state of undifferentiated myoblasts and dissociation of HDAC1 from MyoD during myogenesis.
Creatine Kinase, MM Form ; Gene Expression ; Histone Deacetylase 1 ; Histone Deacetylases ; Histones ; Luciferases ; Muscle Cells ; Muscle Development ; Myoblasts ; Myoblasts, Skeletal ; Sumoylation ; Transfection

BACKGROUNDRetroviral vectors have been widely used to introduce foreign into various target cells in vitro, thus showing relatively high systemic delivery efficiency of various transgene products. The authors investigated the stability and efficiency of skeletal muscle-specific hybrid retroviral vectors in expression of human factor IX (FIX) in vitro and iv vivo.

METHODSFIX cDNA in LIXSN vector was replaced with a FIX minigene containing splicing donor and splicing acceptor sequence of first intron of human FIX gene. Two copies of muscle creatine kinase enhancer (MCK, Me2) were inserted in forward or reverse orientation at NheI site of 3' long terminal repeat (LTR), resulting in two hybrid vectors, which were designated as LMe2IXm2SN(F) and LMe2IXm2SN(R), respectively. The vectors were tested in vitro and in vivo for stability and muscle-specificity of factor IX expression with SCID mice.

RESULTSMuscle cells carrying vector with Me2 expressed significantly higher levels of FIX (up to 1800 ng/106.24 h) than those without Me2, thus suggesting that Me2 could specifically increase expression level of FIX in muscle cells. Myoblasts transduced with LMe2IXm2SN(R) produced much less FIX in vivo in SCID mice than LMe2IXm2SN(F). One or two copies of Me2 sequence were deleted in myoblasts transduced with LMe2IXm2SN(R) without changing the orientation of Me2.

CONCLUSIONSLTR inserted with MCK enhancers can specifically increase human FIX expression in skeletal muscle cells in vitro and in vivo, and MCK enhancer should be positioned in the same orientation as that of LTR promoter.

Animals ; Creatine Kinase ; genetics ; Creatine Kinase, MM Form ; Enhancer Elements, Genetic ; Factor IX ; analysis ; genetics ; Gene Expression ; physiology ; Genetic Techniques ; Genetic Vectors ; Hybridization, Genetic ; Isoenzymes ; genetics ; Mice ; Mice, SCID ; Retroviridae ; genetics ; Terminal Repeat Sequences