We studied the influence of the first intron and 3'-regulatory region of ovalbumin gene (ov) on oviduct-specific transgene expression. The 3'-regulatory region in the oviduct-specific expression vector containing human tissue kallikrein (hK1) cDNA was replaced with bovine growth hormone (BGH) poly A, and the first intron was deleted by restriction enzyme digestion, resulting in five new vectors pOV2K, pOV3K, pOV4K, pOV5K and pOV6K. After mixing with polyethylenimine, we injected same copies of the five vectors via wing vein route into laying hens and compared their expression levels by quantitative assay for enzymatic activities in the egg whites. Among the five vectors tested, the pOV2K containing both the 5'- and 3'-regulatory regions expressed highest level of rhK1 activity, followed by pOV3K with the 3'-regulatory region replaced with BGH poly A, and then by the first intron-shortened vectors pOV4K, pOV5K and pOV6K. These data suggest that both the first intron and 3'-regulatory region of ov gene have enhancing effect on transgene expression in oviduct cells, which should be included in oviduct-specific expression vectors.
Animals ; Animals, Genetically Modified ; Cattle ; Chickens ; Cloning, Molecular ; Female ; Gene Transfer Techniques ; Growth Hormone ; genetics ; Humans ; Introns ; genetics ; Ovalbumin ; genetics ; Oviducts ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Regulatory Sequences, Nucleic Acid ; genetics ; Tissue Kallikreins ; genetics ; Transgenes ; genetics

Lysosomal alpha-mannosidase (EC 3.2.1.24) is a major exoglycosidase in the glycoprotein degradation pathway. A deficiency of this enzyme causes the lysosomal storage disease, alpha-mannosidosis, which has been described in humans, cattle, domestic cats and guinea pigs. Recently, great progress has been made in studying the enzyme and its deficiency. This includes cloning of the gene encoding the enzyme, characterization of mutations related to the disease, establishment of valuable animal models, and encouraging results from bone marrow transplantation experiments.
Animal ; Cats ; Cattle ; Cloning, Molecular ; Disease Models, Animal ; Guinea Pigs ; Human ; Lysosomes/*enzymology ; Mannosidases/*deficiency/*genetics/metabolism ; Mannosidosis/diagnosis/*etiology/*therapy ; Mutation ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic

Lysosomal alpha-mannosidase (EC 3.2.1.24) is an exoglycosidase in the glycoprotein degradation pathway and is encoded by a 3.0 kb cDNA. A 2.3 kb cDNA from a minor species of HeLa cell mRNA was discovered by RT-PCR cloning. Southern blotting and PCR analysis of the HeLa cell genomic DNA showed that the 2.3 kb message was encoded by the lysosomal alpha-mannosidase gene. Sequence comparison of the cDNA with the corresponding genomic DNA indicated that the 2.3 kb message was generated by an unusual intra-exonic joining event.
*Alternative Splicing ; Base Sequence ; DNA, Complementary/genetics ; Exons ; Hela Cells ; Human ; Lysosomes/*enzymology ; Mannosidases/*genetics ; Molecular Sequence Data ; RNA, Messenger/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Support, U.S. Gov't, P.H.S.

Following Escherichia coli lysis with alkali, cetyltrimethylammonium bromide (CTAB) was directly titrated into the supernatant. An easy and feasible technology for plasmid purification was established with the optimized proportion between the quantity of CTAB and plasmid, combined with the specific solution for DNA release and TritonX-114 for endotoxin removal. Quality detection showed that the purified plasmid was free of contamination of host RNA. The host bacterial genomic DNA, endotoxin and bacterial protein were less than 10 microg, 50 EU and 10 microg per mg plasmids, respectively. The ratio of OD260/OD280 was between 1.75-1.85. Eighty percent of the prepared plasmids were presented in the supercoiled form. The plasmid purified with this technology can satisfy all criteria stipulated by FDA. The main advantages of the technology include the avoidance of animal-derived enzymes such as ribonucleases A, Proteinase K and toxic reagents like chloroform and phenol. In addition, the technology has low cost and no pollution.
Cetrimonium Compounds ; chemistry ; Chemical Precipitation ; DNA ; genetics ; isolation & purification ; DNA, Bacterial ; genetics ; isolation & purification ; Escherichia coli ; chemistry ; genetics ; Plasmids ; genetics ; isolation & purification

To evaluate the feasibility of treating hypertension by human tissue kallikrein gene (KLK1) delivery and by enzyme (rK1) administration, two recombinant vectors expressing KLK1 cDNA were constructed for gene delivery (pcDNA-KLK1) and recombinant enzyme preparation (pOV-KLK1). Expression of the pcDNA-KLK1 vector in COS-1 cells was confirmed by immunofluorescence and in spontaneous hypertension rats (SHR) by enzymatic detection. Following intramuscular or intravenous injection with the pcDNA-KLK1 vector, systolic pressure of SHR was significantly decreased, which lasted for 20 d to two months depending on dose, route and/or time of injection. Egg white containing recombinant hK1 was prepared by injection of egg-laying hens with the oviduct-specific expression vector pOV-KLK1 and administered into SHR via oral gavage. Following administration, systolic pressure of the SHR was decreased to that of normal rats, which lasted for 3-5 d depending on the dosage used. These data suggest that both hKLK1 gene delivery and recombinant enzyme administration can be used as alternative strategies for treating human hypertension.
Animals ; Blood Pressure/physiology ; COS Cells ; Cercopithecus aethiops ; Chickens ; Female ; Gene Therapy ; *Gene Transfer Techniques ; Genetic Vectors/genetics/metabolism ; Humans ; Hypertension/genetics/*therapy ; Hypotension/genetics/*metabolism ; Rats ; Rats, Inbred SHR ; Recombinant Proteins/administration & dosage/genetics/metabolism/*therapeutic use ; Tissue Kallikreins/*genetics/metabolism

Objective To establish a rat model of spinal root avulsion and to validate the model by brain-derived neurotrophic factor(BDNF)treatment. Methods To evaluate the motor neuron loss,5 male SD rats were used to undergo spinal root avulsion surgery. One week later, the number of motor neurons in the ventral horn of the spinal cord was as-sessed by histopathology using immunohistochemical staining with a choline acetyl transferase(ChAT)antibody. After this pilot study,40 male SD rats at 7 weeks of age were randomly divided into 4 groups:two control groups,BDNF preventive and treatment groups. Results All rats recovered well post-surgery and no obvious abnormality was observed. Compared with the contralateral side,the number of motor neurons in the ipsilateral avulsed side was significantly decreased at one week after surgery(20.06%,P<0.05). Compared with the control group,there was a significant increase in ChAT posi-tive neurons in the BDNF preventive group(17.85% vs. 93.06%,P<0.0001)or BDNF treatment group(1week after surgery)(26.94% vs. 86.87%, P<0.0001), indicating that the motor neurons were effectively protected by BDNF. Conclusions A rat model of spinal root avulsion is successfully established,which can be valuable for studies of amyotro-phic lateral sclerosis and drug discovery efforts.