Transgenic female mice producing trans 10, cis 12-conjugated linoleic acid present excessive prostaglandin E2, adrenaline, corticosterone, glucagon, and FGF21.

Dietary trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) is a potential candidate in anti-obesity trials. A transgenic mouse was previously successfully established to determine the anti-obesity properties of t10c12-CLA in male mice that could produce endogenous t10c12-CLA. To test whether there is a different impact of t10c12-CLA on lipid metabolism in both sexes, this study investigated the adiposity and metabolic profiles of female Pai mice that exhibited a dose-dependent expression of foreign Pai gene and a shift of t10c12-CLA content in tested tissues. Compared to their gender-match wild-type littermates, Pai mice had no fat reduction but exhibited enhanced lipolysis and thermogenesis by phosphorylated hormone-sensitive lipase and up-regulating uncoupling proteins in brown adipose tissue. Simultaneously, Pai mice showed hepatic steatosis and hypertriglyceridemia by decreasing gene expression involved in lipid and glucose metabolism. Further investigations revealed that t10c10-CLA induced excessive prostaglandin E2, adrenaline, corticosterone, glucagon and inflammatory factors in a dose-dependent manner, resulting in less heat release and oxygen consumption in Pai mice. Moreover, fibroblast growth factor 21 overproduction only in monoallelic Pai/wt mice indicates that it was sensitive to low doses of t10c12-CLA. These results suggest that chronic t10c12-CLA has system-wide effects on female health via synergistic actions of various hormones.

Transgenic mice producing the trans 10, cis 12-conjugated linoleic acid present reduced adiposity and increased thermogenesis and fibroblast growth factor 21 (FGF21).

Trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) from ruminant-derived foodstuffs can induce body fat loss after oral administration. In the current study, a transgenic mouse that produced t10c12-CLA had been generated by inserting the Propionibacterium acnes isomerase (Pai) expression cassette into the Rosa26 locus, and its male offspring were used to elucidate the enduring influence of t10c12-CLA on overall health. Compared to their wild-type (wt) C57BL/6J littermates, both biallelic Pai/Pai and monoallelic Pai/wt mice exhibited reduced plasma triglycerides levels, and Pai/wt mice exclusively showed increased serum fibroblast growth factor 21. Further analysis of Pai/Pai mice found a decrease in white fat and an increase in brown fat, with more heat release and less physical activity. Analysis of Pai/Pai brown adipose tissues revealed that hyperthermia was associated with the over-expression of carnitine palmitoyltransferase 1B, uncoupling proteins 1 and 2. These findings suggest that the systemic and long-term impact of t10c12-CLA on obesity might be mediated through the pathway of fibroblast growth factor 21 when low doses are administered or through enhanced thermogenesis of brown adipose tissues when high doses are employed.

A single amino acid substitution in the FAD-binding domain causes the inactivation of Propionibacterium Acnes isomerase.

We previously demonstrated the efficient production of trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) in Lactococcus lactis by ectopically expressing a Propionibacterium acnes isomerase (pai) gene and also mentioned that a recombinant strain was unable to accumulate t10c12-CLA product, despite the normal transcription. Here, the molecular analysis indicated that this mutated strain harbors a pai gene with a single-nucleotide mutation converting GC50A to GTA, leading to a corresponding change of Alanine residue into Valine. The expression of the reverse mutation resulted in the recovery for enzyme activity. Site-directed mutagenesis indicated that the codon usage of Val17 was not responsible for the enzyme inactivation in the Ala17Val mutation. Western blot analysis revealed that the recombinant PAI protein was not detectable in the His tag-marked Ala17Val mutant. It is, therefore, reasonable to assume that Ala17 residue is critical for PAI functionality.Abbreviations: pai: propionibacterium acnes isomerase; CLA: conjugated linoleic acid; t10c12-CLA: trans 10, cis 12-CLA; LA: linoleic acid (18:2n-6); FAD: flavin adenine dinucleotide.

CTP synthase forms cytoophidia in the cytoplasm and nucleus.

CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase.

Decreased saturated fatty acids, total cholesterol and LDL-C in sdd17 mice.

SDD17, a delta-15 desaturase from the fungus Saprolegnia can convert arachidonic acid to eicosapentanoic acid in yeast, plant embryos, and mammalian cells. Here, we generated transgenic mice that carried two copies of codon-optimized sdd17 cDNA within a non-coding domain of chromosome 6. RT-PCR analysis revealed that the foreign gene was expressed in the transgenic tissues. Gas chromatography showed that the levels of total unsaturated fatty acids in muscle, liver, and spleen tissues were significantly (p<0.05) increased in transgenic mice compared to non-transgenic mice at 3 or 8 weeks of age. In addition, the serum concentrations of total cholesterol and low-density lipoprotein cholesterol in transgenic females, but not in males, were significantly lower than those in sex-matched non-transgenic mice. These results suggest that endogenous sdd17 expression is beneficial for mammalian health and that its effects on fatty acid profiles may differ between sexes.

Mechanism of random integration of foreign DNA in transgenic mice.

Little is known about how foreign DNA is randomly integrated into chromosomes in transgenic animals. In the current study, the insertion sites of 36 transgenic mice were mapped by thermal asymmetric interlaced PCR, and 38 junction sequences were obtained from 30 samples. Analysis of the 38 sequences revealed that 44.7 % of integration events occurred within host gene regions, including 13.2 % (5/38) in exonic regions and 31.6 % (12/38) in intronic regions. The results also revealed that all non-end side integrations of foreign DNA were mediated by short sequence homologies (microhomologies) and that the end side integrations occurred in the presence or absence of microhomologies. In addition, microhomology-mediated mechanisms were also confirmed in four transgenic Arabidopsis thaliana lines. The results indicate that foreign DNA is easily integrated into host gene regions. These results also suggest that the integration of both ends of foreign DNA follows the above-mentioned mechanism in many transgenic/transformed organisms.

Field evaluation of juvenile in vitro embryo transfer (JIVET) in sheep.

The practicality of using juvenile in vitro embryo transfer (JIVET) on a field scale in China was evaluated in each of three seasons (summer, autumn and winter) from 2006 to 2007. A total of 102 donor Merino lambs (18 summer, 69 autumn and 15 winter) aged 4-8 weeks were stimulated with 4 x 40 mg FSH administered at 12h intervals plus 400 IU PMSG given at the time of the first FSH treatment. Overall, 89.2% (91/102) of the lambs exhibited follicle development and 79.1+/-65.5 (mean+/-S.D.) cumulus-oocyte complexes were recovered per donor lamb. Compared with the groups of summer (84.9+/-55.3) and autumn (83.6+/-70.8) lambs, the number of recovered cumulus-oocyte complexes was significantly decreased in winter (51.4+/-43.7; p<0.05). After recovery, the cumulus-oocyte complexes were matured and fertilized in vitro using frozen-thawed semen and culture in synthetic oviduct fluid medium to the 2-4-c stage of development, when they were transferred surgically in groups of 3-8 (5.33+/-1.47) to the ipsilateral uterine horn of a total of 603 synchronized recipients. The overall mean proportion of cumulus-oocyte complexes developing to 2-c embryos was 61.4% (4308/7013) and differed significantly between seasons (summer 38.5%, autumn 66.1%, winter 74.6%; p<0.01). Pregnancy rate assessed by ultrasound examination approximately 60 days after embryo transfer was 54.4% (328/603) overall, and 36.7% (221/603) of the recipients maintained their pregnancy to full-term, producing an average 1.49 (330/221) offspring, of which 1.21 (267/221) were viable and healthy lambs, per pregnant recipient. Pregnancy rate at day 60 was affected by season (summer 40.5%, autumn 56.7%, winter 55.7%; p<0.05), but did not differ significantly between seasons at full-term (summer 34.2%, autumn 38.9%, winter 30.4%; p>0.05). Based on the number of donors stimulated, the total number of offspring and viable progeny produced per donor lamb in autumn (5.81 and 4.87) was significantly (p<0.01) higher than that of summer (2.79 and 1.94) and winter (4.24 and 3.31). This study showed that each donor lamb after stimulation produced an average of 48.6 transferable embryos that resulted in 4.04 viable and healthy progeny. These results indicate that JIVET is a cost-effective method of multiplying desirable sheep genotypes in China.

[Transgenic mice produced by intracytoplasmic sperm injection].

In our previous study, normal and fertile mice were successful produced from oocytes following intracytoplasmic sperm injection (ICSI). In the present study, the possibility of producing transgenic embryos and offspring with this procedure was evaluated. After freezing-thawed once using HEPES-CZB medium without cryoprotectants, the cauda sperm from KM fertile male were exposed to the circular or linear pEGFP-N1 DNA for 1 min and then co-injected into metaphase II oocytes of B6D2F1 strain. When the zygotes with two pronuclei were cultured in CZB medium to day 3.5, 39.1% (9/23) of them, derived from oocytes co-injected with sperm head and pEGFP-N1 plasmid DNA, were expressed GFP protein. After transfer of the ICSI embryos with two pronuclei from co-injection of sperm head and foreign DNA, seven recipients delivered 30 pups (23.8%, 30/126). Southern blot results revealed that three of sixteen offspring integrated with GFP and neomycin genes together (18.8 %). Interestingly, all of them were produced from oocytes co-injected sperm head and linear DNA (33.3%, 3/9), while none of seven ICSI offspring integrated either GFP or neomycin gene in the group of co-injection of sperm head and circular plasmid DNA. These results indicated that the high efficiency of transgenic mouse could be produced by ICSI. It may be shown that linear DNA is more easily to integrate into host genome than circular DNA when ICSI was used to produce transgenic animals.

[Normal mice derived from oocytes following intracytoplasmic sperm injection (ICSI)].

This paper describes the use of piezo-driven micropipette for intracytoplasmic sperm injection of mice eggs. The head of fresh spermatozoa from KM (Kunming) fertile mice was individually injected into mature oocytes of hybrid mice B6D2F1. Approximately eighty three percent of sperm-injected oocytes survived, and 84.0% of them fertilized normally (extrusion of the second polar body and formation of male and female pronuclei). The eggs fertilized by sperm injection could develop in vitro to 2-cell (98% vs 94.7%), 4-cell (89.5% vs 92.1%) stages, no significantly (P > 0.05) different from embryos fertilized in vivo but there were significantly (P < 0.01) few morulae (63.8% vs 84.2%) and blastocysts (25.7% vs 68.4%) developed in vitro after further culture in vitro in the group of ICSI. When 120 embryos at the pronuclear stage were transferred to seven pseudopregnant KM female, 23.3% of the embryos (0 - 50%, depending on the host) reached the full term. Except for three that were cannibalized soon after birth, all of the young (25 pups) developed into normal and fertile adult. Here we report the first birth of mouse offspring following ICSI in China. These studies may increase understanding of the fertilization process and of how ICSI works.

[Molecular cloning, expression mutation of myostatin and study on biochemical activity of its C-terminal peptide].

Myostatin, a member of the TGF-beta family, negatively regulates skeletal muscle development. Mutation of myostatin activity leads to increases muscle growth and carcass lean yield. The bovine myostatin mutation cDNA was amplified by polymerase chain reaction, and then sub-cloned into the expression vector pET-30a( + ) to form the expression plasmid pET30a (+)-action/ Myostatin. The recombinant plasmid was transformed into E. coli BL21. The overexpression product of pET30a (+)-action/ Myostatin was been showed in vitro. Sheep skeletal muscle cell were cultured with the purified myostatin mutation C-terminal peptide. The results of this study suggest that had a powerful activity to stimulate the hyperplasia and proliferation of sheep muscle cells and shows high biochemical activity.

[Transgenic animals bioreactors].

The production of human recombinant proteins in milk of transgenic farm animals offers a safe, very cost-effective source of commercially important proteins that cannot be produced as efficiently in adequate quantities by other methods. This review has summarized the current status of gene selection, vector construct, transgenic methods, economics, and obvious potential in transgenic animals bioreactors. Recently, a more powerful approach was adopted in the transgenic animals founded on the application of nuclear transfer. As we will illustrate, this strategy presents a breakthrough in the overall efficiency of generating transgenic farm animals, product consistency, and time of product development. The successful adaptation of Cre-/lox P-mediated site-specific DNA recombination systems in farm animals will offer unprecedented possibilities for generating transgenic animals.

[Sheep transgenic embryos produced by intracytoplasmic sperm injection].

In this study, the possibility of sheep transgenesis by intracytoplasmic sperm injection (ICSI) was assessed. In experiment 1, activation of ovine oocytes matured in vitro in preparation for ICSI has been investigated with 3.42 mmol/L Ca2+ treatment, ionomycin alone and ionomycin followed by 6-dimethylaminopurine (DMAP) after 3-h delay (group 1, 2 and 3, respectively). After activation, the oocytes were then cultured in SOFaaBSA medium. Cleavage rates were significantly (P<0.05) different among three groups (18.4%, 91.8% and 71.7%, respectively). In additional culture, no parthenotes in group 1, whereas 11% and 17.4% in group 2 and 3 developed to the blastocyst stage. Therefore we used the third activation method in the following ICSI tests. In experiment 2, development of ovine oocytes after ICSI was investigated. Thawed semen from two rams was separated by Percoll centrifugation and was used for ICSI or in vitro fertilization (IVF) trails. A total of 71.8% of oocytes reached the 2-cell stage following living sperm injection, which was significantly (p>0.05) different from those following IVF (41.4%) and sham-ICSI (30.2%). After seven days' culture, no sham-injected oocytes developed into the blastocyst stage, although 7% in ICSI and 16.1% in IVF-oocytes developed into the blastocyst stage, but there was no significant difference in ICSI and IVF groups (p>0.05). In the further study, the possibility of sheep transgenesis by ICSI was assessed. After coinjection of ovine oocytes matured in vitro with dead sperm cold to -20 degrees C and exogenous DNA encoding green fluorescent protein (GFP), seventy-three percent of coinjected oocytes developed to 2-cell stage (33/45) and two of them were transgene-expressing embryos. Among ten embryos at the 16-cell stage, all embryonic cells in one transgenic embryo still expressed GFP. Four coinjected blastocysts were thawed and transferred to the uterine of the two progesterone-synchronized recipient ewe. No pregnancies were detected on the 60th day. These results suggested sheep transgenic embryos could be produced by ICSI and further studies should be performed.