Myostatin, a member of the transforming growth factor beta (TGF-beta) family, is a negative regulator for muscle growth. Loss of the function of this gene is associated with the phenotype described as "double muscling", an extreme form of muscle development characterized by a large increase in muscle mass. Two replacement vectors, pA2T-Mstn4.0 and pA2T-Mstn3.2, were constructed, linearized, and transfected into the bovine fetal fibroblasts through electroporation. 170 drug-resistant cell colonies were obtained in cell culture medium containing 600 microg/mL G418 and 50 nmol/L GCV. Targeted homologous integration occurred in colony No. 58 as identified by PCR, and the targeted colony was further confirmed by sequencing and Southern blotting. This suggested that one allele of myostatin was successfully mutagenized in bovine fetal fibroblasts.
Animals ; Base Sequence ; Cattle ; China ; Electroporation ; Fetus ; Fibroblasts ; cytology ; metabolism ; Gene Targeting ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; veterinary ; Myostatin ; genetics

In producing transgenic livestock, selectable marker genes (SMGs) are usually used to screen transgenic cells from numerous normal cells. That results in SMGs integrating into the genome and transmitting to offspring. In fact, SMGs could dramatically affect gene regulation at integration sites and also make the safety evaluation of transgenic animals complicated. In order to determine the deletion time and methods in the process of producing transgenic goats, the feasibility of deleting SMGs was explored by Cre/LoxP before or after somatic cell cloning. In addition, we compared the efficiency of protein transduction with plasmids co-transduction. We could delete 43.9% SMGs after screening out the transgenic cell clones, but these cells could not be applied to somatic cells cloning because of serious aging after two gene modifications. The SMG-free cells suitable for nuclear transfer were accessible by using the cells of transgenic goats, but this approach was more time consuming. Finally, we found that the Cre plasmid could delete SMGs with an efficiency of 7.81%, but about 30% in SMG-free cells had sequences of Cre plasmid. Compared with Cre plasmid, the integration of new exogenous gene could be avoided by TAT-CRE protein transduction, and the deletion rate of TAT-CRE transduction was between 43.9 and 72.8%. Therefore, TAT-Cre transduction could be an effective method for deleting selectable marker genes.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Organism ; veterinary ; Gene Knockout Techniques ; Gene Targeting ; methods ; Genes, Reporter ; Genetic Engineering ; Genetic Vectors ; genetics ; Goats ; genetics ; Integrases ; chemistry ; metabolism ; Recombination, Genetic ; Transgenes ; genetics

The targeted RNA recombination was attempted to substitute the membrane (M) protein gene and part of the nucleocapsid (N) protein gene of mouse hepatitis virus with the corresponding sequences from bovine coronavirus. Using a defective interfering (DI) RNA-like cDNA construct derived from pMH54, 690 nucleotides representing the entire M gene and the 5' most 915 nucleotides of the N gene of the mouse hepatitis virus Albany 4 mutant were attempted to be replaced. Upon infection of cells with Albany 4 followed by transfection with synthetic RNA transcribed from the DI-like cDNA construct, recombinant mouse hepatitis viruses as the large plaque forming phenotype were isolated by plaque assays at the non-permissive temperature of 391 degrees C. By RT-PCR and sequencing, those large plaque phenotypes were confirmed to have contained the thermostable phenotype marker derived from the transfected RNA, demonstrating that recombination occurred between the Albany 4 genomic RNA and the in vitro RNA transcripts. Further analysis of the recombinant viruses indicated that there combination had taken place within the region of 222 nucleotides between positions 916 and 1,137 of the N gene. This is the region immediately downstream of the replacement sequence and the start of the temperature resistant phenotype marker. The results suggest that the M and part of the N genes of bovine coronavirus may not be able to complement the function of those of mouse hepatitis virus. This study redirects our current approach of utilizing the MHV targeted RNA recombination as a means to study bovine coronavirus genetics towards the construction of an infectious cDNA clone.
Amino Acid Sequence ; Animals ; Base Sequence ; Cattle ; Cells, Cultured ; Coronavirus, Bovine/*genetics ; DNA, Complementary/genetics ; Gene Targeting/veterinary ; Genetic Vectors ; Mice ; Molecular Sequence Data ; Murine hepatitis virus/*genetics ; Nucleocapsid Proteins/*genetics ; Phenotype ; Plaque Assay/veterinary ; RNA, Viral/chemistry/*genetics/isolation&purification ; Reverse Transcriptase Polymerase Chain Reaction/methods/veterinary ; Sequence Homology, Amino Acid ; Transfection/veterinary ; Viral Matrix Proteins/*genetics