| Polyunsaturated fatty acids (PUFAs) have been broadly investigated and have been paid much attention to by the public. PUFAs mainly includes two groups,ω-3 PUFAs andω-6 PUFAs, each also includes many fatty acids from 18 carbons to 22 carbons with two or more unsaturated bonds in their chains. More and more studies shows that PUFAs have broad biological functions, serving as structural components of membrane phospholipids and as precursors of the eicosanoids of signaling molecules. They closely related with many human being's diseases, so their proper levels of content in human bodies is very important for human health. As a whole,ω-6 PUFAs is not crucial for human beings. Contrarily, their excess will bring many healthy problems. Butω-3 PUFAs are beneficial PUFAs, which have been proved to exert many preventive and therapeutic actions on many diseases such as cardiovascular diseases, arthritis, cancer and neuropathic diseases. Human and mammals can not synthesizeω-3 PUFAs in their bodies(so as toω-6 PUFAs), so the PUFA need is, to a great extent, dependent on dietary intake. However the source ofω-6 PUFAs is abundant but that ofω-3 PUFAs is very limited, which lead to the level ofω-3 PUFAs in mammals is relatively too low compared withω-6 PUFAs, the ratio ofω-6/ω-3 PUFAs as high as 16 or more. Therefore, the only thing need to solve is to increase the level ofω-3 PUFAs content in human's rational utilizing of fatty acids, and how to obtain abundant source ofω-3 PUFAs is a focus in the world. Production ofω-3 PUFAs in transgenic animals by gene engineering is becoming the direction for researchers.ω-3 fatty acids desaturase play important role in the synthesis ofω-3 PUFAs , which desaturateω-6 PUFAs into theω-3 PUFA. In recent years, researchers cloned a lot ofω-3 fatty acids desaturase gene from many kinds of organisms. But most of this genes only can synthesize 18-carbonω-3 PUFAs from 18 carbonω-6 PUFAs , so they seems to have no great value of applications. A few genes reported can desaturase longer carbonω-6 PUFAs intoω-3 PUFAs, such as a fungusω-3 desaturase gene from Saprolegnia diclina, desaturates 20-carbonω-6 PUFAs, another fungusω-3desaturase gene from Mortierella alpina 1S-4, desaturates both 18-carbon and 20-carbonω-6 PUFAs. However, these genes have not yet been reported to generate transgenic animals for productionω-3 PUFAs. The only gene used in transgenic animals generation is fat-1 gene, anω-3 desaturase gene from Caenorhabditis elegans, which have been transferred into mammalian cells and mice and as a result, produced abundantω-3 PUFAs, from 18-carbon to 22-carbon. Data show that this gene have great potential for productionω-3 PUFAs. But the level of 22-carbonω-3 PUFAs produced from fat-1 transgenic animals is relatively very low. So the genes that can produce high level of 22-carbonω-3 PUFAs in transgenic animals will be much better for this fields of research.From GenBank search and the published articles, we found that the gene sequence from the worm C.Briggsae (as a result of gene sequencing, the GenBank accession number is CAAC01000009 ) is similar to fat-1 gene, with two amino acids missing compared to the latter. This gene sequence alignment with fat-1 gene revealed 71.5 % gene sequence identity and 86.1% amino acids similarity, and the further study of this gene is not reported. This study established a new method, which was referred to as PCR-restrict enzyme ligation method, to synthesize this Caenorhabditis briggssae gene .By this method, the gene (named as sFat-1) is successfully synthesized following 3 rounds of PCR (7 reactions) and 2 rounds of restrict enzyme ligation (3 reactions). In order to express this worm gene in mammals, its codons were modified and optimized for mammalian gene expression.The synthesized gene was constructed as a mammal expression vector pcDNA3.1-sFat1-EGFP. This vector was introduced into CHO cells by lipid-mediated transfection, and it's expression quickly and effectively elevate the cellularω-3 PUFA contents and dramatically balance the ratio ofω-6/ω-3 PUFAs as GC-MS analysis of cellular lipids extracted from stably selected cells showed. The amount of totalω-6 PUFAs dropped from 48.97% (in GFP cells)to 35.29%(in sFat-1 transferred cells),whereas the amount of totalω-3 PUFAs increased from 7.86% to 24.02% respectively. Theω-6/ω-3 ratio also dropped from 6.23 to 1.47 accordingly. These data demonstrates the Caenorhabditis briggssae sFat1 was synthesized successfully and can be used for further study. What is important is that this gene transferred into CHO cells produced higher level of DPA (5.98%) and DHA(1.97%) compared to the former similar research with fat-1 gene.Then we use vector pcDNA3.1-sFat1-EGFP as transgenic vector to produce transgenic mice. The DNA was microinjected into mouse zygote and transferred into recipients, at last 33 pups born. Analysis by PCR and southern blot, five of them are transgenic. GC-MS analysis results show that many tissues of these transgenic mice produced all kinds ofω-3 PUFAs, such as LA(18:3n-3),EPA(20:5n-3),DPA(22:5n-3) and DHA(22:6n-3), which similar with that of in sFat-1 gene transferred CHO cells. But these mice produced higher level of DHA and DPA. For example, the tissue of the transgenic mice muscle produced 12.5% of DHA( averagely) and 23.04% of totalω-3 PUFAs, all of which far exceed the former studies ofω-3 desaturase transgenic anmals. Our research also produced transgenic mice for mammary expression of the sFat-1 gene by microinjecting the constructed vector of pBC1-sFat1, 5 from 24 born pups are transgenic. GC-MS analysis results show that only 1 of them expressed the sFat-1 gene in the milk (other tissues not detected).Moreover, the expression level slightly higher than that in the milk of pcDNA3.1-sFat1-EGFP transgenic mice. This shows it is possible for mammary expression of the sFat-1 gene in domestic animals to produceω-3 PUFAs.Lentiviral vectors are high efficient vectors for gene transfer. In order to investigate their potential for production of transgenic animals, we constructed a lentiviral vector for the expression of sFat-1,a gene from C.Briggsae encodingω-3 Fatty Acid Desaturase. By co-transfection of pLP1,pLP2,pLP/VSVG with this vector, 293 FT cells produced lentiviral particle, which can infect mammalian cells(tested by NIH3T3 cell line). The tilter estimated to about 5.0×106TU/ml. Also, the lentiviral particle proved can infect embryos when co-cultured with mouse embryos. This research means a further step to production lentiviral transgenic animal expression a functional gene for actual use.
|
PDF Full Text Request