Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two(P<0.05).The development rate of embryos reconstruct-ed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization(FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that(1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA genotype through FISH.

Effects of in Vitro Maturation Time of Oocytes on Sheep Nuclear Transfer Efficiency

[Objective] The study aimed to provide references for the time of oocyte maturation in vitro and enucleation in the course of sheep nuclear transfer(NT).[Method] Compared the effects of different maturation time of oocytes on enucleation efficiency and reconstructed embryo development by means of blind enucleation and fluorescence microscopy.[Result] Treatment of IVM(in vitro maturation)19-21 h was significantly higher than IVM 16-18 h treatment in oocyte maturation rate(P<0.05)and was significantly higher than IVM 22-24 h treatment in enucleation rate(P<0.05).Three treatments had no significant difference in cleavage rate and blastocyst rate(P>0.05),but IVM 19-21 h treatment was significantly higher than the other 2 treatments in average cell number of blastocysts(P<0.05).[Conclusion] The appropriate in vitro maturation time of oocytes was 19-21 h for sheep nuclear transfer,which could significantly improve the quality of blastocysts according to the cell number per blastocyst(P<0.05).

Pregnancies Resulting from Transgenic Embryos with Human Lactoferrin Gene Produced by Somatic Cell Nuclear Transfer

[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.

Human Pro-insulin Transgenic Calf Derived from Somatic Cell Nuclear Transfer

The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r） gene and enhanced green fluorescent protein （EGFP） gene linked through an inner ribosomal entry site （IRES） sequence directed by a Cytomegalovirus （CMV） promoter, was used for enrichment and selection of the transgenic cells and preimplantation embryos. Transgenes were introduced into bovine fetal fibroblast cells （BFF） cultured in vitro through electroporation （900 V/cm, 5 ms）. Transgenic bovine fibroblast cells （TBF） were enriched through addition of G418 in culture medium （800 μg/mL）. Before being used as a nuclear donor, the TBF cells were either cultured in normal conditions （10% FBS） or treated with serum starvation （0.5% FBS for 2-4 days） followed by 10 hours recovery for G1 phase synchronization. Transgenic cloned embryos were produced through GFP-expressing cell selection and SCNT. The results were the percentage of blastocyst development following SCNT was lower using TBF than BFF cells （23.2% VS 35.2%, P 〈 0.05）. No difference in the percentage of cloned blastocysts between the two groups of transgenic nuclear donor of normal and starvation cultures were observed （23.2% VS 18.9%, P 〉 0.05）. Two to four GFP-expressing blastocysts were transferred into the uterus of each synchronised recipient. One pregnancy from of seven recipients （21 embryos） was confirmed by rectum palpation 60 days after embryo transfer and one recipient has given birth to a calf at term. PCR and DNA sequencing analysis confirmed that the calf was produced using human proinsulin transgenic animal.

Development in vitro in Reconstitution of Embryo from Porcine Somatic Cell Nuclear Transfer

In present paper,a study on reconstitution of porcine oocytes by using nuclear transfer with cumulus cells(CC) and fibroblast cells(FC) was carried out.Reconstituted oocytes which were the fusion with CC and showed a cleavage rate of 56.7%,developed into morula (11.7%) and blastocysts (6.7%) phases which were higher than those derived from the fusion with FC( P <0.05).The results of this study also involved the effects of oocyte collection method,activation protocol and maturational age of recipient oocytes during the in vitro develpoment of nuclear transfer embryos which were reconstituted with cultured cumulus cells.The cumulus cells synchronized in G 0/G 1 phases through serum starvation culture,were transferred into enuclated oocytes which were collected by aspiration or dissection method and cultured for 33 or 44 h.Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulsation and 6 DMAP,and cultured for 6 days.As for the oocyte collection methods,activation treatment in the presence of cytochalasin B and activation protocols did not affect the developmental rate of embryos reconstituted with 44 h mature recipients.However,the development rate of reconstituted embryos with 33 h mature recipients were significantly higher( P <0.05) by activation with the combination of electric pulsation and 6 DMAP.These results suggest that the reconstituted porcine embryos derived from cultured cumulus cells can develop into the blastocyst stage and that the development of the former could be improved for the reconstitution with young oocyte cytoplast after the activation with the combination of electric pulsation and 6 DMAP.

An Improved Enucleation Method of Bovine Somatic Cell Nuclear Transfer

The aim of this work was to improve the rate of conventionally blind enucleation for bovine somatic cell nuclear transfer. The cross section of a 0.5 ml Eppendorf tube was attached with a sheet of 400 mesh/inch2-cell screen after the bottom of the Eppendorf tube had been cut, and put into a 1 mL Eppendorf tube. In experiment 1, the oocytes in the metaphase Ⅱ stage were placed on the membrane in the Eppendorf tube, and centrifuged at 1,000, 2,000, or 3,000 r/min for 10 min, respectively. The oocytes were stained with Hoechst 33342 and then the relative position of the first polar body to the chromosomes, and the efficiency of enucleation were evaluated. In experiment 2, enucleated oocytes were fused with granulosa cells, following centrifugation and enucleation, and the potential development of the reconstituted embryos was estimated. The results indicated that the rate of enucleation in oocytes after centrifugation at 2,000 r/min for 10 min was 86.6% with an angle less then 20° between the first polar body and chromosomes. The rate of enucleation in cells spun at 2,000 r/min was higher than that of controls （87.4% vs. 64.4%, P 〈 0.05）. Fur- thermore; centrifugation of recipient oocytes did not have a detrimental effect on the development of reconstituted embryos following nuclear transfer. In conclusion, centrifugation assisted enucleation may significantly improve the rate of bovine oocyte enucleation.

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN m RNA and magnetic beads with somatic cell nuclear transfer

The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout （KO） cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase （GGTA1） gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 （IB4） lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.

Multinucleon transfer dynamics in nearly symmetric nuclear reactions

Within the framework of the dinuclear system model,the multinucleon transfer dynamics for nearly symmetric nuclear collisions has been investigated.The reaction mechanism in the systems of 198Pt+198Pt and 204Hg+198Pt was investigated at beam energies around the Coulomb barrier.It was found that the isotopic yields are enhanced with increased incident energy in the domain of proton-rich nuclides.However,the production on the neutron-rich side weakly depends on the energy.The angular distribution with the beam energy was also analyzed in the multinucleon transfer reactions.Projectile-like fragments were produced toward the forward emission with increasing incident energy.The target-like fragments manifested the opposite trend in the transfer reactions.

Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model

This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.

Establishment of customized mouse stem cell lines by sequential nuclear transfer

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from nuclear transfer （NT） embryos, may play a major role in the new era of regenerative medicine. In this study we established forty nuclear transfer-ESC （NTESC） lines that were derived from NT embryos of different donor cell types or passages. We found that NT-ESCs were capable of forming embryoid bodies. In addition, NT-ESCs expressed pluripotency stem cell markers in vitro and could differentiate into embryonic tissues in vivo. NT embryos from early passage RI donor cells were able to form full term developed pups, whereas those from late passage RI ES donor cells lost the potential for reprogramming that is essential for live birth. We subsequently established sequential NT-RI-ESC lines that were developed from NT blastocyst of late passage R 1 ESC donors. However, these NT-R I-ESC lines, when used as nuclear transfer donors at their early passages, failed to result in live pups. This indicates that the therapeutic cloning process using sequential NT-ESCs may not rescue the developmental deficiencies that resided in previous donor generations.

Whole-genome methylation analysis reveals epigenetic variation between wild-type and nontransgenic cloned,ASMT transgenic cloned dairy goats generated by the somatic cell nuclear transfer

Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.

Production of homeobox A10 gene transgenic pigs by somatic cell nuclear transfer

Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover, Hoxa10 plays a key role in regulating the embryo implantation of sows. This study aimed to generate transgenic pigs using Hoxa10 via somatic cell nuclear transfer(SCNT). We established seven Hoxa10-transgenic cell lines, and two of the cell lines were selected as nuclear donors for the transfer. A total of 1 270 cloned embryos were generated and transferred to five surrogate mothers(Landrace×Yorkshire). Eight cloned male piglets were produced including one with cryptorchidism. Six transgenic piglets grew up healthy and produced 56 offspring. Finally, we obtained six transgenic male pigs and 26 transgenic positive offspring that can be used to further study the regulatory mechanism of Hoxa10 on the reproductive performance of pigs.

Characterization of 14 anonymous nuclear loci in Pinus thunbergii and their cross-species transferability

We characterized 14 anonymous nuclear loci from Pinus thunbergii Parl., an important pine species native to Japan. One hundred and twenty-six single nucleotide polymorphisms （SNPs） were identified from these loci, giving a frequency of 1 SNP per 51 bp. Nucleotide di- versity （0） ranged from 1.06 × 10^-3 to 11.87 × 10^-3, with all average of 4.99 × 10^-3. Only one locus （mK45） deviated significantly from the Hardy-Weinberg equilibrium. Thirteen of 14 loci were applicable in other pine species. These loci will be useful for nucleotide variation studies and will provide material for SNP-based marker development in P. thun- bergii and related species.

Factors Affecting the Efficiency of Embryonic Cell Nuclear Transfer in Rabbits

Factors affecting the efficiency of nuclear transfer （NT） in rabbits were examined in the present study. When 100 V mm of pulse strength and 15 us of pulse duration were employed, 3 and 4 electronic pulses resulted in significantly more cytoplasts fused with donor cells compared with 2 electronic pulses （P〈 0.05）, but no significant difference was found in the cleavage rate of reconstructed embryos among the three groups （P〉0.05）. When the duration and number of electronic pulse were fixed at 15 ps and 3 times, increase of pulse intensity from 100 V mm 1 to 150 V mm^-1 and 200 V mm^-1 resulted in a significantly decrease in the cleavage rate of reconstructed embryos （P〈 0.05）, although the fusion rate did not significantly differ among the three groups （P〉 0.05）. Significantly more reconstructed embryos cleaved and developed to blastocysts when they were derived from donor embryos at the 8-16-cell stage, in comparison with the reconstructed embryos derived from donor embryos at the compact morula stage （P 〈 0.05）, although the fusion rate was similar （P 〉 0.05）. Activation of cytoplasts prior to fusion increased the cleavage rate （P〈 0.05） and blastocyst development （P〈 0.05） of reconstructed embryos, but decreased the fusion rate （P 〈 0.05） compared with cytoplasts activated post fusion. More reconstructed embryos developed to blastocysts when they were cultured in TCM ＋ 3% OCS at the first 48 h and then cultured in TCM199＋ 10% FCS, in comparison with the reconstructed embryos cultured in either TCM199＋ 10% FCS or TCM199＋3% OCS （P 〈 0.05）. When 22 NT embryos were transferred into the oviducts of one recipient rabbit, one recipient rabbit delivered a female rabbit at 34 days of gestation. In conclusion, either electrofusion parameter or developmental stage of donor embryos have a significant effect on the efficiency of NT, NT embryos require different concentration of serum at their different development stages.

Studies on Recipient Rabbit Oocytes in Nuclear Transfer

Studies were carried out to find out the optimal parameters of enucleation and electroactivation ofrecipient rabbit oocyte for successful nuclear transfer,using the fluorescent stain,DAPI(4-6-diamidino-2-phenylindole),and electroactivation.According to the position of metaphasechromosomes in relation to the first polar body,the oocytes were classified into three types:1.thosewith chromosomes juxtaposed to the polar body;2.those with chromosomes in its adjacency; and3.those with chromosomes further removed.The relative proportions of each type appeared to varywith the time of maturation at which the oocytes were collected,with those of the later typesincreasing as the maturation process went further on.In addition,in-vitro cultivation ofelectroactivated oocytes gave the best results with oocytes that matured in-vito after injection ofovulating hormones(LH or HCG)and oocytes that were cultivated in-vitro for 17-19 hours.As aresult,it is recommended that oocytes be selected from those collected from the oviducts 13-15 hoursafter injection of LH or HCG,and electrofusion and electroactivation be done aftermicromanipulation and in-vito cultivation for 2-4 hours.By so doing,it is expected to achieve thehighest enucleation rate of oocytes and the highest fusion rate,the highest activation rate and thehighest development rate of the restructured embryos.

Serial Nuclear Transfer of Goat-Rabbit Interspecies Reconstructed Embryos

The experiments of serial nuclear transfer were conducted between Boer goat and rabbit. The enucleated oocytes of rabbit were used as recipients while the blastomeres of goat morula was used as nuclear donor. The reconstructed embryos developing to morula were used as donor for serial cloning. As a result, two generations of reconstructed embryos were obtained, including 58 first generation reconstructed embryos and 14 second generation reconstructed embryos. The fusion rates were 79.5 and 70%, respectively, and there was no significant difference between them （P〉0.05）. The cleavage rates were 75.9 and 28.6% respectively with significant difference （P〈0.01）. No blastocyst was obtained from the second generation reconstructed embryos while 13.8% of first generation reconstructed embryos developed to blastocyst.

Aberrant DNA methylation in 5′ regions of DNA methyltransferase genes in aborted bovine clones

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases （DNMTs）, therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5＇ regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmtl and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones （AF1, AF2 and AF3） showed either hypermethylation or hypomethylation in the 5＇ regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized （IVF） fetuses. Besides, we found that the 5＇ regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5＇ regions is probably associated with the high abortion of bovine clones.

Nusselt number correlation for turbulent heat transfer of helium-xenon gas mixtures

A gas-cooled nuclear reactor combined with a Brayton cycle shows promise as a technology for highpower space nuclear power systems.Generally,a helium-xenon gas mixture with a molecular weight of14.5-40.0 g/mol is adopted as the working fluid to reduce the mass and volume of the turbomachinery.The Prandtl number for helium-xenon mixtures with this recommended mixing ratio may be as low as 0.2.As the convective heat transfer is closely related to the Prandtl number,different heat transfer correlations are often needed for fluids with various Prandtl numbers.Previous studies have established heat transfer correlations for fluids with medium-high Prandtl numbers(such as air and water)and extremely lowPrandtl fluids(such as liquid metals);however,these correlations cannot be directly recommended for such helium-xenon mixtures without verification.This study initially assessed the applicability of existing Nusselt number correlations,finding that the selected correlations are unsuitable for helium-xenon mixtures.To establish a more general heat transfer correlation,a theoretical derivation was conducted using the turbulent boundary layer theory.Numerical simulations of turbulent heat transfer for helium-xenon mixtures were carried out using Ansys Fluent.Based on simulated results,the parameters in the derived heat transfer correlation are determined.It is found that calculations using the new correlation were in good agreement with the experimental data,verifying its applicability to the turbulent heat transfer for helium-xenon mixtures.The effect of variable gas properties on turbulent heat transfer was also analyzed,and a modified heat transfer correlation with the temperature ratio was established.Based on the working conditions adopted in this study,the numerical error of the property-variable heat transfer correlation was almost within 10%.

Numerical Research of Heat Transfer of Supercritical COR2R in Channels

With the worldwide development of nuclear power plant and requirement of saving energy and resource, high thermal efficiency and economical competitiveness are achieved by using supercritical COR2R with spe-cial thermal properties and better flow and heat transfer characters. In this paper, heat transfer of supercritical COR2R has been investigated in square and triangle array tube-bundle of cooled system in reactor using com-putational fluid dynamics (CFD) code FLUENT, and the basic knowledge of heat transfer of supercritical COR2R and first experience of CFD simulation are obtained. The effect of mesh structures, turbulence models, as well as flow channel size is analyzed. The choice of turbulence model adopted in simulating supercritical COR2R is recommended. Comparing the effect of heat transfer with supercritical COR2R and supercritical water as cooled medium, the results show that the former was higher. The new idea is provided for choice of cooled medium and improving thermal efficiency this paper.