Search

Klonlama teknolojisi; erişkin bir hücre çekirdeğinin yumurta hücresi içerisine konulup geriye programlanarak embriyonal döneme geri döndürülmesi prensibine dayanmaktadır. Ancak bu geri programlamayı etkileyen faktörler tam olarak aydınlatılamamıştır. Bu nedenle elde edilen klonlar ve klonların yavrularının daha detaylı bir şekilde tanımlanması ile klonlama teknolojisi daha anlaşılır ve kontrol edilebilir hale getirilebilir. Bu çalışmanın amacı bir ırkın bireylerinin farklı bir ırka ait yumurta kaynağı kullanılarak klonlanması sonucu elde edilen klonların normal bir ırk populasyonu oluşturmasının mümkün olup olmadığını ortaya koymak adına klon ve jenerasyonlarını moleküler olarak karakterize etmek ve böylece ileride bu teknolojinin nesli tükenmiş bir ırkın geriye getirilmesindeki muhtemel potansiyelini daha iyi anlamaya çalışmaktır. Bu amaçla çalışmada daha önce TÜBİTAK- TOVAG-104O360- projesi ile üretilmiş 5 klon boz sığır (1 erkek, 4 dişi) ve bu klonların yavruları (2 erkek, 4 dişi) materyal olarak kullanılmıştır. Öncelikle klonlar, onların yavruları ve klonların üretilmesinde kullanılan verici hücreden elde edilen genomik DNA’larda 10 mikrosatellit belirteç kullanılarak klonların genomik DNA açısından verici hücrelerin birebir kopyası olduğu ve yavrularında bu klonlara ait olduğu teyit edilmiş ve mtDNA D-loop bölgesi dizi analizi ile de klonların mtDNA’larının yumurta kaynaklı olduğu ve dolayısıyla verici hücreden farklı olduğu tespit edilmiştir. Ayrıca bu farklı mtDNA varlığı klonların yavrularında da izlenmiştir. Cloning technology is based on the principle of turning back to embryonic period by reprogramming of that an adult cell nucleus was put into the oocyte cell. However, affecting factors of this reprogramming has not been clarified completely. Therefore, cloning technology can be made more understandable and controllable with a more detailed manner identifying of obtained clones and their offspring. The purpose of the study is to characterize the clones and their generations molecularly to reveal whether it is possible or not that creation of a normal race population through clones which acquired as a result of cloning individuals of one race by using ovular source which belongs to a different race, and thus to try to understand better that possible potential of this technology to reinstate an extincted race. For this purpose, 5 clone grey cattles (1 male, 4 female ) and their offsprings ( 2 male, 4 female ) which generated with TUBİTAK TOVAG-104O360- project had been used as material. First of all 10 microsatellite markers were used to test the clones, their offspring and donor cells which were used for producing the clones.It has been confirmed that the clones are the copy of donor cells and all offspring belong to the clones and by sequencing of D-Loop region of mtDNA, it has been determined that the origin of mtDNA from clones are eggs and therefore are different than donor cell. In addition the presence of mtDNA difference has been observed in offspring of the clones.

Klonlama teknolojisi; erişkin bir hücre çekirdeğinin yumurta hücresi içerisine konulup geriye programlanarak embriyonal döneme geri döndürülmesi prensibine dayanmaktadır. Ancak bu geri programlamayı etkileyen faktörler tam olarak aydınlatılamamıştır. Bu nedenle elde edilen klonlar ve klonların yavrularının daha detaylı bir şekilde tanımlanması ile klonlama teknolojisi daha anlaşılır ve kontrol edilebilir hale getirilebilir. Bu çalışmanın amacı da bir ırkın bireylerinin farklı bir ırka ait yumurta kaynağı kullanılarak klonlanması sonucu elde edilen klonların normal bir ırk populasyonu oluşturmasının mümkün olup olmadığını ortaya koymak adına klon ve jenerasyonlarını moleküler olarak karakterize etmek ve böylece ileride bu teknolojinin nesli tükenmiş bir ırkın geriye getirilmesindeki muhtemel potansiyelini daha iyi anlamaya çalışmaktır. Bu amaçla çalışmada daha önce TÜBİTAK- TOVAG-104O360- projesi ile üretilmiş 5 klon boz sığır (1 erkek, 4 dişi) ve bu klonların yavruları (2 erkek, 4 dişi) materyal olarak kullanılmıştır. Öncelikle klonlar, onların yavruları ve klonların üretilmesinde kullanılan verici hücreden elde edilen genomik DNA’larda 10 mikrosatellit belirteç kullanılarak klonların genomik DNA açısından verici hücrelerin birebir kopyası olduğu ve yavrularında bu klonlara ait olduğu teyit edilmiş ve mtDNA D-loop bölgesi dizi analizi ile de klonların mtDNA’larının yumurta kaynaklı olduğu ve dolayısıyla verici hücreden farklı olduğu tespit edilmiştir. Ayrıca bu farklı mtDNA varlığı klonların yavrularında da izlenmiştir.

Yeryüzünün en önemli gen merkezlerinden birinde yer alan Türkiye’nin zengin biyolojik çeşitliliğini korumak ve gerektiğinde kullanmak zorunluluk olarak kabul edilmelidir. Çünkü biyolojik çeşitlilik ekonomik ve genetik zenginliğin bir göstergesi olup, tıp, tarım, gıda güvencesi ve endüstride önemli yararlar sağlamaktadır. Çiftlik hayvanları genetik kaynakları, biyolojik çeşitliliğin önemli bir parçasını oluşturmaktadır. Türkiye'de hayvan genetik kaynakları yeterince değerlendirilememiş, hatta bazıları daha tam olarak tanımlanmadan yok olmuş veya yok olma tehlikesi ile karşı karşıya kalmıştır. Bazı ırklar bilimsel kriterlere göre risk sınırının dışında kabul edilmekle birlikte, azalma hızının çok yüksek olduğu ırklarda mevcut sayının risk sınırına, hatta bu sınırın da altına kısa sürede düşeceği beklenmektedir. Bu durum; ekonomik, sosyal ve çevresel gelişmelerin hayvancılıkta farklı yerlerde çeşitli düzeylerde olmak üzere entansifleşmeyi zorunlu kılması, az girdi ile yetiştirilebilen, buna karşılık düşük verimli olan yerli ırkların yerini hızlı bir şekilde kültür ırkları veya melezlerinin almasının sonucudur. Oysa yerli ırklar yüzyıllardır yetiştirilegeldikleri çevrenin kendine özgü koşullarına çok iyi uyum sağlamış, verimleri düşük olsa bile özgün nitelikleri olan, dayanıklı, kanaatkar, yetersiz çevre koşullarında üreyebilen hayvanlardan oluşur. Yerli ırkların yok olması, taşıdıkları bu ayırıcı özelliklerin de yok olması anlamına gelmektedir. Gelecekte bu özelliklerin hangisine gereksinme duyulacağını şimdiden tahmin etmek güç veya olanaksızdır. Kaldı ki bu gen kaynaklarının bugün saptanmamış olan olası özellikleri de ancak bunların varlıklarını sürdürebilmesi halinde elde tutulabilir. Belirtilen noktalardan hareketle; bildiride Türkiye Yerli Koyun Irklarının korunmasının gerekliliği, korumaya alınacak ırkların belirlenmesi, koruma yöntemleri, Türkiye koyun ırklarının mevcut durumu, bunların korunmasına yönelik çabalar, korumayla ilgili olarak karşılaşılan sorunlar ve alınması gereken önlemler üzerinde durulmaktadır

This study was performed to examine whether adult fibroblast cells can be manipulated genetically and whether clonal lines derived from those cells can support embryonic development. A primary adult cell line was established from the ear of an aged cow. Adult cells with a plasmid containing the enhancer green fluorescence protein (EGFP) gene, and non-transfected cells were used for cloning. Green fluorescence expression was observed in 35/49 (71.4%) adult clones. The developmental rates of embryos were significantly lower for cell lines expressing EGFP (11.4%) than for non-transfected cells (20.1%, P < 0.05). However, there was no decrease in nuclear transfer (NT) developmental rates (21.5%) when donor nuclei from EGFP transfected cell lines not expressing EGFP but retaining neomycin-resistance gene expression were used as donor nuclei. The NT embryos from transfected and non-transfected cell lines had similar morphology and cell numbers. The results indicated that adult cells can complete clonal propagation including transfection and selection and can be used to produce transgenic NT embryos.

This study was performed to determine the capability of embryonic stem (ES) cells at late passage number to form functional germ cells and to orientate the sexual development of chimeric mice produced from ES cell injection into embryos. When an R1 ES cell line was routinely cultured on mitotically inactivated feeder cells in culture medium supplemented with leukemia inhibitory factor (LIF), the colonies maintained an undifferentiated ideal ES cell morphology. When the ES cells were injected into 45 eight-cell embryos, 38 embryos (84%) combined with ES cells. Twenty-eight percent of the embryos transferred to recipients developed to term. Thirty percent of offspring born were chimeric. In these chimeric individuals the contribution of ES cells to the coat was about 70% and 99% in two male and one female chimeras, respectively. Furthermore, ES cells contributed to the germ line in the female chimera whereas they did not contribute to the germ line in two male chimeras. This study showed that female chimeras may result from injected embryos with the male ES cell and may transmit the ES cell genome to their offspring.

Vitrification is becoming a preferred method for pre-implantation embryo cryopreservation. The objective of this study was to determine the differentially expressed genes of in vivo- and in vitro-produced bovine embryos after vitrification. In vitro- (IVF) and in vivo-derived (IVV) bovine blastocysts were identified as follows: in vitro-produced fresh (IVF-F), in vitro-produced vitrified (IVF-V), in vivo-derived fresh (IVV-F), in vivo-derived vitrified (IVV-V). The microarray results showed that 53 genes were differentially regulated between IVF and IVV, and 121 genes were differentially regulated between fresh and vitrified blastocysts (P < 0.05). There were 6, 268, 962, and 17 differentially regulated genes between IVF-F × IVV-F, IVF-V × IVV-V, IVF-F × IVF-V, and IVV-F × IVV-V, respectively (P < 0.05). While gene expression was significantly different between fresh and vitrified IVF blastocysts (P < 0.05), it was similar between fresh and vitrified IVV blastocysts. Significantly up-regulated KEGG pathways included ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis in the fresh IVF blastocyst samples, while sphingolipid and purine metabolisms were up-regulated in the vitrified IVF blastocyst. The results showed that in vitro bovine blastocyst production protocols used in this study caused no major gene expression differences compared to those of in vivo-produced blastocysts. After vitrification, however, in vitro-produced blastocysts showed major gene expression differences compared to in vivo blastocysts. This study suggests that in vitro-produced embryos are of comparable quality to their in vivo counterparts. Vitrification of in vitro blastocysts, on the other hand, causes significant up-regulation of genes that are involved in stress responses.

Vitrification is becoming a preferred method for pre-implantation embryo cryopreservation. The objective of this study was to determine the differentially expressed genes of in vivo- and in vitro-produced bovine embryos after vitrification. In vitro- (IVF) and in vivo-derived (IVV) bovine blastocysts were identified as follows: in vitro-produced fresh (IVF-F), in vitro-produced vitrified (IVF-V), in vivo-derived fresh (IVV-F), in vivo-derived vitrified (IVV-V). The microarray results showed that 53 genes were differentially regulated between IVF and IVV, and 121 genes were differentially regulated between fresh and vitrified blastocysts (P?

Öz bulunamadı.

The aim of this study was to compare Solid Surface Vitrification (SSV) technique and classic vitrification technique in in vitro produced 8 days old bovine blastocysts. Cryopreservation of mammalian embryo has great importance for genetic resources conservation, embryo transfer, veterinary and clinical reproductive biotechnology and animal assisted reproductive technologies. Immature oocytes were maturated then fertilized with frozen-thawed bull semen and cultured until blastocyst stage in commercial sequential culture medium for 8 days. Blastocysts were vitrified in two different groups as SSV and classic vitrification and non-vitrified blastocysts were used as control group. After vitrification, vitrified blastocysts were warmed and cultured for 1 day. For this aim, blastocyst viability rate and median cell number were investigated. The blastocyst viability rate that vitrified by classic vitrification (34.8%) were found to be lower than those vitrified by SSV (82.6%1 and control group blastocysts (100%). However, median cell numbers of vitrified-warmed blastocysts were found higher in SSV (124) than classic vitrification (104). Median cell number of control group was detected as 213. As a result blastocyst viability rate and median cell number in SSV group was higher than classic vitrification group, there was a significant difference between SSV and classic vitrification group (p

The researchers investigated the effect of blastocyst development and quality of blastomere aspiration techniques on eight-cell mouse embryos. The results clearly indicate that the in vitro development of biopsied mouse embryos depended on the suitable aspiration method. This method related to preimplantation genetic diagnosis for the genesis of animal models for animal disorders from embryos. In this study, female CB6 F1 (Balb/cXC57b1/j) hybrid mice were superovulated with hormones and superovulated females were sacrified approximately 68 h after hCG administration. About eight-cell embryos were recovered from oviducts of sacrified mouse in M2 medium. Before biopsy, all embryos were incubated to decrease cell to cell contacts to microdrops of Ca2+/Mg2+ free QAM HTF (3 mg mL(-1) BSA Fraction V) with HEPES for 90 min at 37 degrees C. A single blastomere of eight-cell embryos were aspirated by the aspiration pippetes under inverted microscope (4X). After biyopsy, embryos were cultured in SAGE medium supplemented with 5% CO2, 5% O-2 and 90% air at 37 degrees C for up to expanded blastocyst stage. After culture, 267 biopsied embryos out of 234 (88.32 +/- 7.5%) expanded blastocysts developed from the biopsy group and total cell number mean 67.8 +/- 17.2% were recorded in the experiment group and 126 non-biopsied embryos out of 118 (94.4 +/- 7.78%) expanded blastocysts developed from control embryos and total cell number mean 70 +/- 15.4% was recorded in the control group. In the present study, there was no difference in blastocysts developmental rates at post biopsy group and control group (p = 0.05). In conclusion, the biopsy the method described here is an optimal method of blastomere aspiration on in vivo mouse embryos.

BAGIS/0000-0002-1140-8058 WOS: 000265889100028 In vitro embryo culture is an important step of in vitro production of bovine embryos. The aim of this study was to compare the in vitro development of bovine embryos in G1.3/G2.3 sequential culture media and CR1aa culture medium. Oocytes were obtained from ovaries of slaughtered cows and matured in TCM-199 medium in humidified air containing 5% CO(2) at 39 degrees C. Matured oocytes were fertilized in vitro using frozen bull sperm prepared with Percoll separation. After fertilization, the presumptive zygotes were denuded from cumulus cells and randomly allotted for 2 treatments: Culture in CR1aa (n = 116) and G1.3/G2.3 (n = 125). The embryo culture was carried out in 50 mu L droplets of the media that were placed in incubator containing 6% CO(2) 5% O(2) and 89% N(2). The embryos were evaluated on days 3, 5 and 7 following in vitro fertilization. The difference of cleavage rates between 2 treatments was not found significant (p>0.05). However, significant difference was observed between 2 treatments at the level of the obtaining rate of morula and blastocyst (p

Yeryüzünün en önemli gen merkezlerinden birinde yer alan Türkiye’nin zengin biyolojik çeşitliliğini korumak ve gerektiğinde kullanmak zorunluluk olarak kabul edilmelidir. Çünkü biyolojik çeşitlilik ekonomik ve genetik zenginliğin bir göstergesi olup, tıp, tarım, gıda güvencesi ve endüstride önemli yararlar sağlamaktadır. Çiftlik hayvanları genetik kaynakları, biyolojik çeşitliliğin önemli bir parçasını oluşturmaktadır. Türkiye'de hayvan genetik kaynakları yeterince değerlendirilememiş, hatta bazıları daha tam olarak tanımlanmadan yok olmuş veya yok olma tehlikesi ile karşı karşıya kalmıştır. Bazı ırklar bilimsel kriterlere göre risk sınırının dışında kabul edilmekle birlikte, azalma hızının çok yüksek olduğu ırklarda mevcut sayının risk sınırına, hatta bu sınırın da altına kısa sürede düşeceği beklenmektedir. Bu durum; ekonomik, sosyal ve çevresel gelişmelerin hayvancılıkta farklı yerlerde çeşitli düzeylerde olmak üzere entansifleşmeyi zorunlu kılması, az girdi ile yetiştirilebilen, buna karşılık düşük verimli olan yerli ırkların yerini hızlı bir şekilde kültür ırkları veya melezlerinin almasının sonucudur. Oysa yerli ırklar yüzyıllardır yetiştirilegeldikleri çevrenin kendine özgü koşullarına çok iyi uyum sağlamış, verimleri düşük olsa bile özgün nitelikleri olan, dayanıklı, kanaatkar, yetersiz çevre koşullarında üreyebilen hayvanlardan oluşur. Yerli ırkların yok olması, taşıdıkları bu ayırıcı özelliklerin de yok olması anlamına gelmektedir. Gelecekte bu özelliklerin hangisine gereksinme duyulacağını şimdiden tahmin etmek güç veya olanaksızdır. Kaldı ki bu gen kaynaklarının bugün saptanmamış olan olası özellikleri de ancak bunların varlıklarını sürdürebilmesi halinde elde tutulabilir. Belirtilen noktalardan hareketle; bildiride Türkiye Yerli Koyun Irklarının korunmasının gerekliliği, korumaya alınacak ırkların belirlenmesi, koruma yöntemleri, Türkiye koyun ırklarının mevcut durumu, bunların korunmasına yönelik çabalar, korumayla ilgili olarak karşılaşılan sorunlar ve alınması gereken önlemler üzerinde durulmaktadır. Since Turkey has been complimented as one of the gene centers of the world, the biological diversity of Turkey has to be preserved to be be used when necessary. Biological diversity indicates the economical and genetical wealth, and it ensures important benefits in medicine, agriculture industry and food safety Farm animal genetic resources are the important part of the biological diversity. Animal genetic resources of Turkey have not been adequately evaluated, even some of genotypes extinct or facing extinction risk. Although some of the native breeds of Turkey are not at stake of extinction risk by the scientific criteria, some breeds have already decreased in numbers so close to extinction in a short time because, despite their some superior characteristics, culture breeds or crosses replace native breeds due to the economical, social and environmental changes and overall development in the country. Extinction of native breeds leads to the loss of superior characteristics such as endurance, resistance, modesty and reproduction ability under insufficient environmental conditions that native breeds posses in their homelands. Which of these characteristics can be useful in the future can not be easily predicted. Therefore, the probable characteristics of the genotypes can be conserved by their existence. On this basis; necesity of protection of native sheep breeds of Turkey, determination of breeds for protection, protection methods, present situation of the sheep breeds of Turkey, protection studies and efforts, problems and cautions of the protection studies of Turkey are discussed in this communication note.

WOS: 000253792400008, PubMed ID: 18444564, The studies for the development of transgenic mice models which provide important profits for the studies concerning immunopathogenesis of hepatitis B virus (HBV) infections are in progress since 20 years. For this purpose different lineages bearing whole HBV genome or selected viral genes have been developed and their usage in clarifying the HBV replication and pathogenesis mechanisms have been emphasized. The aim of this study was to develop and breed a HBV carrier mice model. In the study the full HBV genome has been transferred to mouse embryos by microinjection procedure. Following transgenic manipulation, the HBV carriers among the daughter mice have been detected by molecular methods in which HBV-DNA replication and expression have been shown. The manipulations for transgene transfers have been performed in TUBITAK Marmara Research Center Transgene Laboratory, Gebze, istanbul. The HBV-DNA carrier mice have been demonstrated by polymerase chain reaction (PCR) using the DNA samples obtained from tail tissues and also by dot-blot hybridization of the mice sera. Integrated HBV-DNA has been detected by applying in-situ hybridization to the liver tissue sections. HBV-DNA expression has been shown by reverse transcriptase PCR method with total RNA molecules that have been isolated from the liver tissues of the HBV-DNA carrier mice. HBsAg has been detected in the liver by immunohistochemical method, and HBsAg and HBeAg have additionally been demonstrated by ELISA. HBV genome, expression of the genome and the expression products have been determined in approximately 10% of the mice of which HBV-DNA have been transferred. By inbreeding heterozygote carrier mice, homozygote HBV transgenic mice line have been obtained. These HBV transgenic mice are the first lineages developed in our country. It is hopefully thought that this HBV carrier transgenic mouse model may contribute to the studies on the pathogenesis of HBV infections which are important health problems in the world as well as in Turkey.

Here we describe the generation of transgenic mice carrying type III fish antifreeze protein (AFP) gene and evaluate whether AFP type III protects transgenic mouse ovaries and testes from hypothermic storage. AFPs exist in many different organisms. In fish, AFPs protect the host from freezing at temperatures below the colligative freezing point by adsorbing to the surface of nucleating ice crystals and inhibiting their growth. The transgenic expression of AFP holds great promise for conferring freeze-resistant plant and animal species. AFP also exhibits a potential for the cryopreservation of tissues and cells. In this study, we have generated 42 founder mice harboring the Newfoundland ocean pout (OP5A) type III AFP transgene and established one transgenic line (the line #6). This study demonstrated that AFP gene construct has been stably transmitted to the mouse progeny in the F3 generations in the line #6. Furthermore, the presence of AFP transcripts was confirmed by RT-PCR analysis on cDNAs from liver, kidney, ovarian, and testis tissues of the mouse from F3 generation in this line. These results indicate that ocean pout type III AFP gene could be integrated and transmitted to the next generation and stably transcribed in transgenic mice. In histological analysis of testis and ovarian tissues of nontransgenic control and AFP transgenic mice it has been shown that both tissues of AFP transgenic mice were protected from hypothermic storage (+4 degrees C). The AFP III transgenic mice obtained for the first time in this study would be useful for investigating the biological functions of AFP in mammalian systems and also its potential role in cryopreservation.

6. ÖZET Bu araştırma 150 IU PMSG ile follikül gelişimi uyarılan tavşanlarda ovulasyonları meydana getirmek amacıyla farklı dozlarda verilen HCG (100, 150 IU) ve GnRH (1.2, 2, 4, 8 mg buserelin)'ın ovulasyon oranı ve embriyo kalitesi üzerine etkilerini araştırmak amacıyla yapıldı. Araştırmada 70 adet dişi, 4 adet erkek tavşan kullanıldı. Çiftleşmeden 72 saat sonra ovaryum reaksiyonlarım gözlemek ve embriyo kazanmak amacıyla yapılan operasyonlar neticesinde ovulasyon oranlan 100 IU HCG uygulanan grupta %49.85, 150 IU HCG uygulanan grupta %60.82 olarak belirlendi. Farklı dozlarda buserelin uygulanan gruplarda ise ovulasyon oranları %42.94, %44.48, %46.96, %64.47 iken kontrol grubunda bu oran %24.44 olarak tespit edildi. Aynı hayvanlarda sağlıklı embriyo oranı sırası ile HCG grubunda %68.75 ve %94.79, GnRH grubunda ise %90.31, %79.74, %87.22, %89.65 olarak bulunurken kontrol grubundan istenilen kalitede embriyo elde edilemedi. 57 7. SUMMARY This research is carried out to investigate the effects of increasing doses of HCG (100,150 IU) and GnRH (1.2,2,4,8 mg) to ovulation rate and embryo quality in the rabbits at which follicle development was stimulated by 150 IU PMSG. 70 female and 4 male rabbits were used in the experiments. By the operations which were performed 72 hrs after mating, ovarium reactions were examined and the embryos were recovered. The ovulation rates were 49.85% in the 100 IU HCG treated group whereas 60.82% in the 150 IU HCG group. The ovulation rates in the groups which treated with increasing doses of GnRH were 42.94%, 44.48%, 46.96%, 64.47% and 24.44% in the control group. The healthy embryo rates in the same animals were 68.75% and 94.79% in the HCG groups, respectively. In the GnRH groups these rates were 90.32%, 79.74%, 87.22%, 89.65% respectively,whereas no healthy embryo was recovered from the control group. 72