Multipotential differentiation and potential applications of adipose-derived stem cells / 生物工程学报

Adipose tissue contains a population of multipotent cells called adipose-derived stem cells (ADSCs). With the similar properties of marrow-derived mesenchymal stem cells, ADSCs have the ability to differentiate differentiate towards adipogenic, osteogenic, chondrogenic, myogenic, endothelial, hematopoietic, hepatic, islet, and neurogenic cell lineages. As adipose tissue in harvested in large amounts with minimal morbidity, it can be widely used in tissue engineering, organ repair and gene therapy. This paper focused on the plasticity of ADSCs and reviewed the new advances of this field. Finally, the problems and prospect for application was also discussed.

The targeting study of human serum albumin gene / 生物工程学报

To generate transgenic porcine which expresses human serum albumin (HSA), the HSA gene targeting vector was constructed with HSA cDNA as the gene of interestand partial porcine serum albumin (PSA) gene as homologous arms which respectively were 7.2 kb 5' regulation sequence and 2.8 kb genomic sequence from the first intron to the fourth intron. The resistant gene neo was inserted into intron 1 and tk was ligated to the 3' end of the construct. Linearized targeting construct DNA was introduced into the fibroblast cells obtained from porcine fetus by electroporation. The positive-negative selection was performed and survival clones were screened by PCR and Southern blot. Three colonies with correct homologous recombination were obtained. Our results set a good basis for the establishment of transgenic porcine by gene target and nuclear transfer methods.

Advance of Ser/Thr Kinase Pololike Kinase 1 (Plk1) Research / 中国生物工程杂志

Pololike kinase 1(Plk1)contain an Nterminal Ser/Thr kinase catalytic domain and a Cterminal region that contains two poloboxes.As a key regulator of multiple steps during cell cycle across eukaryotic species,many proteins interact with Plk1.Plk1 is highly expressed in malignant cells and serves as a negative prognostic marker in specific human cancer types.Plk1 is a potential target for cancer therapy.Some novel smallmolecule inhibitors of pololike kinase 1 provide novel opportunities for cancerdrug discovery,such as BI 2536,ON01910.

The synthesis and function analysis of omega-3 fatty acid desaturase gene from Caenorhabditis briggssae / 生物工程学报

Omega-3 polyunsaturated fatty acids (PUFAs) have been broadly investigated and shown to exert many preventive and therapeutic actions besides their important role in maintenances human health and normal development. In mammals, the level of omega-3 PUFAs is relatively too low compared with omega-6 PUFAs, which metabolically and functionally distinct from omega-3 PUFAs and often have important opposing physiological functions. Either the inefficiency of omega-3 PUFAs or the excess of omega-6 PUFAs will cause many healthy problems. So methods have been sought to increase the amount of omega-3 PUFAs and to improve the omega-6/omega-3 ratio in body. In this study, the sFat-1 gene, which putatively encodes a omega-3 fatty acid desaturase, was chemically synthesized according to the sequence from Caenorhabditis briggssae (with codon usage modified), and constructed into 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 elevated the cellular omega-3 PUFAs (from 18-carbon to 22-carbon) contents and dramatically improved the ratio of omega-6/omega-3 PUFAs. Cellular lipids extracts from stably selected cells were analyzed with GC-MS and the results showed that amount of total omega-6 PUFAs dropped from 48.97% (in GFP cells)to 35.29% (in sFat-1 cells), whereas the amount of total omega-3 PUFAs increased from 7.86% to 24.02%, respectively. The omega-6/omega-3 ratio also dropped from 6.23 to 1.47. These data demonstrates the Caenorhabditis briggssae omega-3 Fatty Acid Desaturase gene, sFat-1, was synthesized successfully and can produce omega-3 PUFAs by using the corresponding omega-6 PUFAs as substrates, which shows its potential for use in the production of omega-3 PUFAs in transgenic animals.

The construction and characterization of human pro-urokinase mutant / 生物工程学报

It is very easy for the pro-UK to lose it's biological activity because of the digestion of pro-UK by the thrombin or the inhibition of pro-UK by the PAI-I. So three pro-UK mutant (pro-UK) genes were constructed in this experiment with the PCR point-mutant method. The thrombin cleavage site Arg156 in pro-UK was mutated into His156, and named as pro-UKM1; PAI binding sites Arg178, Arg179, Arg181 were mutated into Lys178, Lys179, His181, named as pro-UKM2; The mutant containing His156, Lys178, Lys179, His181 as pro-UKM3. Three mutants were expressed in CHO cells respectively and analyzed with SDS-PAGE fibrin plate assay and western blot. The results showed that the three mutants and the native pro-UK have the same single electrophoresis band indicating most of the pro-UK was single chain. In vitro plasma clot lysis assays indicated that the pro-UKM1 have the ability to resistant against thrombin digestion; pro-UK2 could resist against PAI inhibition; while pro-UK3 improved resistances against both thrombin and PAI. It looks very promising that the pro-UK3 can be a new medicine of dissolving thrombus.

The ht-PAm cDNA knock-in the goat beta-casein gene locus / 生物工程学报

The production of recombinant protein is one of the major successes of biotechnology, animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animal mammary gland bioreactor are being used for this purpose. Gene targeting is a more powerful method to produce mammary gland bioreactor, and nuclear transfer from cultured somatic cells provides an wonderful means of cell-mediated transgensis. Here we describe efficient and reproducible gene targeting in goat fetal fibroblasts to place the human tissue plasminogen activator mutant (ht-PAm) cDNA at the beta-casein locus, and would produce the transgenic goat by nuclear transfer. To construct the gene targeting vector pGBC4tPA, the milk goat beta-casein genomic DNA sequence for homologous arms had been cloned firstly. The left arm was 6.3 kb fragment including goat beta-casein gene 5' flanking sequence, and the right arm was 2.4 kb fragement including beta-casein gene from exon 8 to exon 9. The ht-PAm cDNA was subcloned in the goat beta-casein gene exon 2, and the endogenous start condon was replaced by that of ht-PAm. The bacterial neomycin (neo) gene as positive selection marker gene, was placed in the beta-casein gene intron 7, the thymidine kinase (tk) as the negative selection marker gene, was just outside the right arm. The validity of the positive-negative selection vector (PNS), was tested, and targeting homologous recombination (HR) were elevated to 5-fold with the negative selection marker using the drug GANC. The DNA fragment in which two LoxP sequence was delected effectively using Cre recombinase in vitro. Goat fetal fibroblasts were thawed and cultured to subconfluence before transfection, about 10(7) fibroblasts were electoporated at 240V, 600 microF in 0.8 mL PBS buffer containing linear pGBC4tPA. transfected cells were cultured in collagen-coated 96-wellplate for 24h without selection, then added the drug G418 (600 microg/mL) and GANC (2 micromol/L). After 12 days of selection, well separated G418r/GANCr clones were isolated and expanded in 24-wellplate. 244 clones were selected, and only 90 clones could grow and be tested by PCR screening for targeting. The primary result demonstrated that 31 targeting cell clones with homologous recombination events were obtained, and 2 cell clones was verified by DNA sequence analysis on the homologous recombination region.

Production of mammary gland bioreactor by gene targeting of somatic cells / 生物工程学报

Producing mammary gland bioreactor showed great advantage over many years, but the level of transgenic expression was low in transgenic animals and the diversity was more great because of the position effect of transgene and the artificial recombination of the gene elements. Gene targeting based on the principle of gene homologous recombination had been studied and applied, because the transgene could be integrated precisely in the chromosome. This review summary the current status of producing mammary gland bioreactor by the technology of gene targeting and nuclear transfer using the somatic cell lines. These aspects were discussed, including the characteristic and difficulties of gene targeting, the strategies to improve the efficiency of gene targeting, the different features of between the strategy of promoter-trap and the Cre-LoxP system, etc; for the others, how to select the cell lines with the different strategies of gene targeting, how to raise the times of cell lines that was cultured after the gene targeting. Somatic cell nuclear transfer offers new and exciting opportunities in the areas of the gene targeting. However, the field as a whole is still difficult and complex. In this paper, we described recent advances and novel approaches, which resulted in progress during the last year. Key problems hindering further progress are addressed, for example, how to increase the efficiency of nuclear transfer. With the technology of gene targeting and nuclear transfer, it should provide a general way to produce specific genetic changes in several mammalian species. We are clearly at the dawn of a new era in mammalian genetic technology.