Expression Of Lumbrokinase Gene In Goat Mammary Gland And Construction Of Lumbrokinase Transgenic Mice

Lumbrokinase is an active protein - complex from whole earthworm tissue extract, which shows anticoagulative and fibrinolytic activities. In 1991, Mihara et al. have first demonstrated a novel fibrinolytic enzyme extracted from Lumbri-cus rubellus and named it as Lumbrokinase. At present there are many Lumbrokinase preparations, such as Pu - en Fu, Baiao, Boluoke and Thrombolysis capsule, to mainly treat ischemic cerebrovascular disease, cardiovascular disease, and myocardial infarction. In 2001, Fan et al. proved that earthworm fibrinolytic enzyme Ⅲ — 1 isolated from Lumbricus rubellus could be transported into blood through intestinal epithelium and keep its biological function in circulation, which showed that lumbrokinase worth further investigating and exploiting. Now though some lumbrokinase genes have been cloned and sequenced, expressing active protein has not been reported. So expressing the gene and preparing lots of lumbrokinase product by mammary gland bioreactor technique are of importance for research and exploitation.Mammary gland bioreactor technique is one of applications of transgenic animal, which includes establishing transgenic animal by introducing promoter element specially expressing in mammary gland, directing target gene expression in mammary gland cells, and extracting recombinant protein from milk. In 1987, Gordon et al. first obtained human plasminogen activation product from milk of transgenic mice. Further researches demonstrated that mammary gland had abilities to express extensive target genes. Some important proteins for clinical purpose , such as alpha - 1 - antitrypsin, human tissue plasminogen activator, lact-oferrin, and human protein C have been expressed in mammary gland succes-sively and many of products have been used in clinic. The methods of preparing mammary gland bioreactor are same as those of preparing transgenic animals, which include classical microinjection technique, sperm introducing method, re-combinant retrovirus infecting method, and nuclear transplanting technique. Microinjection technique method was mostly used among them, but it had disadvantages of high - expense, time - waste, low transgenic efficiency, and random gene integration. Though nuclear transplant method represents the trend of transgenic animal research, it means strict experimental conditions and considerable laboratorial level, which are difficult to measure up. Retrovirus gene transfer method has been reported to prepare transgenic animals, which is low - cost and needs only common experimental conditions, and should become a practical method. In this article we tried this method and used synthetic lumbrokinase as target gene. It has been proved in preliminary test that lumbrokinase gene could be transiently expressed in goat mammary gland, which to some extent represented expression level of target gene in transgenic animal. We expect that the gene could be expressed in goat milk introduced by recombinant retroviral particles infecting mammary gland cells.For reducing blindness in preparing transgenic big domestic animals, expressing ability of expression element including target gene should be tested before preparation. The methods of screening and testing expression vector include expression in cultured mammary gland cells in vitro, transient expression in mammary gland, and preparing bioreactor model of transgenic mice. The method of eukaryotic cell expression is easy and intuitionistic, but does not entirely show expression features of animal in physiological condition. The method of transient expression in milk is also easy and objective, and can partly show features of expression in physiological condition, however, persistent period of expression is short, and quantity of expression is correlated with that of transfer. So transient expression is often used to generally estimate expressing efficiency of expression vector. Preparation of transgenic mice is time - waste and high — expense, and sometimes does not show expression features of target gene in big transgenic bioreactor, but propagating period of mice is short, fetiferous ability is high, and expression of target gene can be investigated in the whole, so this method ismostly used to value mammary gland specific vector. In this article we prepared lumbrokinase transgenic mice by classical microinjection technique, which grounded for further research of transgenic domestic animals.Materials and Methods1. 1 AnimalsBoer goats and Kunming mice were fed in our Department of Laboratory Animals.1.2 Bacterial strain, Plasmids and Cell linesEscherichia coli. strain DH5ot was stored in our laboratory. Mammalian expression vector pcDNA3 was from Invitrogen Company. Vector pGEMT - bCP containing goat beta - casein gene promoter, pMD18T - LK. containing synthetic lumbrokinase gene cDNA, and recombinant retrovirus vector pLNC — LacZ were constructed in previous experiments and stored. Cell lines PA317 and NIH3T3 were purchased from Institute of Shanghai Cell Research, Scientific Academy of China.1. 3 Transient expression of Lumbrokinase gene in goat mammary gland1.3. 1 Mammary gland expression vector containing goat beta - casein promoter and synthetic lumbrokinase cDNA was contructed and prepared largely by alkaline lysis method.1.3.2 Vector DNA was injected directly into goat mammary gland in stable milk secretion period.1.3.3 Activities of lumbrokinase gene expression were detected by fibrin plate method.1.4 Expression of lumbrokinase gene in goat milk introduced by recombinant retroviral vector1. 4.1 Recombinant retroviral vector containing goat beta - casein promoter and synthetic lumbrokinase cDNA was contructed.1.4. 2 Recombinant vector was packaged by PA317 cell and cloned cell lines were screened by G418.1.4. 3 Integration of target gene in genomic DNA of cloned cell lines was i-dentified by PCR.1.4.4 Titre of recombinant virus was detected by infecting NIH3T3 cell.1.4. 5 Recombinant virus infected goat mammary gland cells by directly injecting into goat mammary gland before production.1.4.6 Activities of lumbrokinase gene expression in goat milk were detected by fibrin plate method.1.4.1 Preparation of lumbrokinase transgenic mice1. 5 Preparation and purification of DNA for microinjection.1.5.1 Preparation of transgenic mice by classical microinjection technique.1.5.2 Detection of transgenic mice and their offspring by PCR and Southern blot.1.5.3 Activities of lumbrokinase gene expression in mouse milk were detected by fibrin plate method.Results2. 1 Transient expression of lumbrokinase gene in goat milkMammary gland expression vector pBLK was constructed containing the goat P - casein promoter and lumbrokinase gene cDNA. Vector pBLK was directly injected into the goat mammary gland and its expression in goat milk was determined by fibrin plate method. Results showed that lumbrokinase gene could transiently be expressed in goat milk. Lumbrokinase activity in 6 -9h after injection reached peak which was 2. 0 x 10 U/L,and expression lasted for 60h. Repeated 3 times injections had no effect on transient expression.2. 2 Expression of lumbrokinase gene in goat milk introduced by recombinant retroviral vectorThe replication - defective retrovirus vector pLNBLK containing the goat (3 - casein promoter and lumbrokinase gene cDNA was constructed. Vector pLN-BLK was transferred into PA317 packaging cell line by lipofectamine method and under the screen of G418 cell clones had been achieved. Results of PCR proved that the target gene had integrated into the genome of cell clones. Titers of recombinant retrovirus ranged from 2 x 104 to 1 x 105CFU/ml detected by infectingNIH3T3 cell line. Virus culture medium 10ml had been directly injected into the goat mammary gland during 2 weeks before production and milk specimens gathered after bearing were detected by fibrin plate method. Results showed that lumbrokinase gene could be expressed in goat milk and activity of lumbrokinase had not changed markedly in the 45th day after production.2. 3 Preparation of lumbrokinase transgenic miceDNA fragment for microinjection containing whole expression elements was gained by digesting pBLK with Sal I and Pvu II. About 800 fertilized eggs of mice were injected using classical microinjection technique, and about 500 living eggs were transplanted into 29 pseudopregnant mice, among which 11 pregnant mice produced 43 offspring. The offspring were identified by PCR and Southern blot, 3 of which were lumbrokinase transgenic mice. Target gene can be transferred by 2 living transgenic mice, which was proved by PCR. The expression of lumbrokinase gene in the milk of transgene mice was detected using fibrin plate mehtod, but fibrinolytic activity could not be found in it.Conclusions3. 1 Mammary gland expression vector containing lumbrokinase gene was constructed and injected directly into goat mammary gland in stable milk secretion period, and target gene could transiently be expressed in milk.3.2 Recombinant retroviral vector containing lumbrokinase gene was constructed, and target gene transferred by packaged virus could be expressed in milk.3. 3 Lumbrokinase transgenic mice were established successfully and transgene can be transferred to offspring normally.