Generation of rat offspring derived from sperm cryopreserved/banked in the National BioResource Project for the rat followed by transportation to another institution

To preserve genetic resources efficiently in rats, sperm cryopreservation is essential. This study aimed to confirm the ability of cryopreserved and transported rat spermatozoa to fertilize through intrauterine insemination. The Komeda miniature rat Ishikawa is a mutant caused by the autosomal recessive mutation mri. The epididymal sperm was frozen with egg yolk medium and banked at the National BioResource Project (NBRP), Kyoto University. The sperm was transported to Azabu University, then thawed at 37℃. The thawed semen was inseminated into the uterine horns of recipients; its motility was around 10%. Seven of 15 inseminated female rats became pregnant and 13 live pups were born. The results indicate that rat spermatozoa cryopreserved at NBRP are capable of restoring genetic resources through intrauterine insemination. We also confirmed the usefulness of assisted reproductive technologies for the rat including sperm cryopreservation and intrauterine insemination

Candida albicans

Genotypes of Candida spp. isolated from exhalation of 20 dolphins, 11 water samples from captive pools, and 24 oral cavities of staff members in an aquarium using a combination of multiple drug resistance 1 gene (MDR1) and the internal transcribed spacer (ITS) 1 5.8s-ITS 2 regions of ribosomal RNA gene (ITS rDNA) sequences were studied. The holding ratios of the dolphins, captive pools, and staff members were 70, 90, and 29%, respectively. Isolated pathogenic yeast species common to the dolphins and environments were Candida albicans and C. tropicalis. Identical genotypes in both Candida spp. based on the combination of MDR1 and ITSrDNA were found in some dolphins, between a dolphin and a staff, among dolphins and environments, and among environments. The results indicated the diffusion and exchange of pathogenic yeasts at the aquarium among dolphins and environments. The isolates at the aquarium showed higher rates of resistance to azole antifungals compared to reference isolates

ビオチン欠乏が及ぼすラット海馬への影響

ビオチン欠乏時および回復後のラット海馬歯状回のニューロジェネシスについて免疫組織化学的に検証した。Wistar系雄ラットを用いた。ビオチン欠乏(BD)群には生理食塩水を，対照(BS)群にはビオチン(200mg/kg)を毎日腹腔内投与し，5週間後に麻酔下で全放血後，脳を摘出し，20μmの連続凍結切片とした。切片は抗BrdU抗体と抗neuronal nuclei(NeuN)抗体の二重免疫染色，一部は抗polysialic acid(PSA)抗体による免疫染色，抗BrdU抗体と抗glial fibrillary acidic protein(GFAP)抗体を用いた二重免疫染色を実施し，海馬のニューロン新生部位である歯状回顆粒細胞層およびその下層(subgranular zone : SGZ)について行い，各染色における陽性細胞数をカウントした。新生ニューロンを示すBrdU/NeuN陽性細胞数とPSA陽性細胞数は，BS I群と比較してBD I群で有意(P<0.001)に減少していた。同様にSGZにおいて神経前駆細胞を示すGFAP/BrdU陽性細胞数についてもBD I群で減少する傾向(P=0.06)を示した。一方，回復期を設けたBD II群・BS II群においては，BrdU/NeuN陽性細胞数は両群間でほぼ同様の値を示し，PSA陽性細胞数はBD II群のほうがBS II群と比べて，増加する傾向(P=0.06)を示した。GFAP/BrdU陽性細胞数はBrdU/NeuNと同様に両群間でほぼ同様の値を示した。以上のことから，ビオチン欠乏状態では海馬歯状回のニューロジェネシスは抑制され，逆にビオチン欠乏状態回復後では対照群のそれと比べて同等のレベルまで改善することが示唆された。We previously confirmed that learning ability of a biotin-deficient (BD) rat decreased and LTP of its hippocampus was suppressed. In this study, the neurogenesis of hippocampal dentate gyruses of rats in BD conditions were investigated immunohistochemically. Wistar strain rats, 3-week-old, were fed BD diet. BD group was subjected to daily i.p. injection of saline for 5 weeks. Control (BS) group received same diet with daily injection of biotin (200,μg/kg, i.p.). The rats of the BD and BS groups were administered 5-Bromo-2\u27-deoxyuridine (BrdU, 50 mg/kg, i.p.) every 5 days, at a total of 4 times before autopsy. After exsanguination under anesthesia, the brains were removed, fixed with 4 % para-formaldehyde, sectioned 20 μm thick, and stained immunohistochemically with anti-neuronal nuclei (NeuN) and anti-BrdU antibodies. The numbers of BrdU positive cells of BD and BS were 39.1 ± 1.0 and 53.0 ± 0.8, respectively, and the numbers of BrdU/NeuN positive cells were 26.8 ± 0.7 and 39.3 ± 0.6, respectively. Both numbers of BrdU and BrdU/NeuN positive cells of the BD group showed significantly small values (p < 0.001). The neurogenesis of the hippocampus was suppressed in the BD condition. This suggests that biotin has an important role in the development and maintenance of brain functions

トランスジェニックマウスを用いたコプラナーPCBsの毒性評価 : 化学物質の安全性評価モデルTgラットの開発

突然変異は,癌や加齢にともなう疾患などの原因と考えられているし,いくつかの環境中の化学物質は,人の体細胞や生殖細胞における突然変異の発生を増加させているとも考えられている。そのため,突然変異を誘発するような化学物質の安全性を評価することは,変異原誘発性や発癌性のフィールドでは特に重要な問題となっている。このことから遺伝子突然変異の有無を効率良く評価できる有用なモデルとしてλEG10DNAを導入したTgマウス(gpt delta)が作出され,生体に対する化学物質の安全性を評価に用いられている。しかし,マウスでは採取できるサンプル量などに限りがあるため,マウスよりも大きいラットでのTg個体の作出が切望されている。本研究はgpt deltaマウスと同じ特性を持つTgラットの作出を試みた。ラットは繁殖効率が高く,世界的に使用頻度が高いWistar系ラットを用いた。大腸菌gpt遺伝子とλファージのred/gam遺伝子から構成されるλEG10DNAをラット受精卵に導入した。これらの胚を偽妊娠誘起したレシピエント雌へ移植し,自然分娩によって産子を得た。得られた産子の尾よりDNAを抽出し,PCR,電気泳動によってλEG10DNAの導入を調べた。導入が確認できた11例の内,12週齢以上に成育した9例をWistar系ラットと交配させ,得られた125例の産子全てにおいてλEG10DNAの導入を調べたところ,56例の個体で確認できた。しかし,これらの個体は表現型が全てヘテロ型であるため,遺伝的に均一なホモ型ではない。今後は56例のヘテロ型の内in vitroパッケージングによるλEG10DNA回収効率の高い個体を選抜し,さらに戻し交配を行うことでホモ型であるTgラットを作出する。Mutations are implicated in the etiology of cancer and other age-related diseases. Some environmental chemicals may increase the occurrence of mutations in human somatic and/or germ cells. Thus, the evaluation of the potential of chemicals to induce mutations is a particularly important issue in the field of environmental mutagenesis and carchinogenesis. Transgenic mice have been produced by introducing λEG10DNA into mice (gpt delta mice), because they are thought to be useful in effectively evaluating the safety of new chemicals in vivo. However, the number of the mice taken as samples is limited, and it is necessary to produce transgenic rats (Tg rats), which are more reproductive and bigger than mice. This study made an attempt to produce Tg rats, which have the same characteristics of gpt delta mice. Wistar rats were used in this study because they have high reproductive efficiency, and are frequently used worldwide. λEG10DNA composed of the Escherichia gpt gene and red/garn gene of λphage was introduced into fertilized eggs of the rats. The embryos were placed in female rat recipients, which had been induced to be pseudopregnant. The recipients had babies by spontaneous delivery. DNA was extracted from the tails of the baby rats to ascertain whether λEG10DNA was introduced in the rats using PCR, and electrophoresis. The introduction was confirmed in eleven baby rats. After that, nine rats out of the eleven, which reached twelve weeks old, were mated with Wistar rats. One hundred twenty-five babies were obtained, and introduction of λEG10DNA was ascertained in fifty-six individuals among the babies. However, all of the fifty-six individuals were of the Hetero phenotype. That is, they were not of the Homo phenotype, which means genetically uniform. We are planning to select individuals with high recovery efficiency of λEG10DNA from the fifty-six individuals by in vitro packaging, and then backcross them in order to produce Homo phenotype Tg rats

トランスジェニックマウスを用いたコプラナーPCBsの毒性評価 : 化学物質の安全性評価モデルTgラットの開発

ラットにgpt遺伝子を導入できたが,gpt遺伝子を含むλEG10DNAは確認できなかった。The evaluation of chemicals which induce mutations is a particularly important issue in the field of environmental mutagenesis and carcinogenesis. Transgenic mouse (gpt delta, mouse) have been produced by introducing lEG10 phage DNA, composed of the Escherichia gpt gene and red/gam gene of lphage, because they are thought to be useful in effectively evaluating the safety of new chemicals in vivo. However, the sample size taken from the mouse is limited. It is necessary to produce transgenic rats, which have the same characteristics of gpt delta mice. We produced 11 transgenic rats as a founder having gpt gene. In the next generation, 56 pups out of a total of 125 also had gpt gene. We examined a collection efficiency of lEG10DNA including gpt gene form their DNA in vitro. 56 rats having gpt gene were used. A small part of liver of in each rat was removed under anesthesia. DNA was extracted form the liver. The DNA collection efficiency of lEG10DNA including gpt gene was examined by in vitro packaging method. As for each rat, it was confirmed that gpt gene was introduced. However, lEG10DNA was collected in neither individual by the packaging method. It may be thought that a little number of the copies of lEG10DNA incorporated into genomic DNA, or that a body portion of 1 phage being broken even if lEG10DNA was introduced

トランスジェニックマウスを用いたコプラナーPCBsの毒性評価 : 化学物質の安全性評価モデルTgラットの開発

突然変異は,癌や加齢にともなう疾患などの原因と考えられているし,いくつかの環境中の化学物質は,人の体細胞や生殖細胞における突然変異の発生を増加させているとも考えられている。そのため,突然変異を誘発するような化学物質の安全性を評価することは,変異原誘発性や発癌性のフィールドでは特に重要な問題となっている。このことから遺伝子突然変異の有無を効率良く評価できる有用なモデルとしてλEG10DNAを導入したTgマウス(gpt delta)が作出され,生体に対する化学物質の安全性を評価に用いられている。しかし,マウスでは採取できるサンプル量などに限りがあるため,マウスよりも大きいラットでのTg個体の作出が切望されている。本研究はgpt deltaマウスと同じ特性を持つTgラットの作出を試みた。ラットは繁殖効率が高く,世界的に使用頻度が高いWistar系ラットを用いた。大腸菌gpt遺伝子とλファージのred/gam遺伝子から構成されるλEG10DNAをラット受精卵に導入した。これらの胚を偽妊娠誘起したレシピエント雌へ移植し,自然分娩によって産子を得た。得られた産子の尾よりDNAを抽出し,PCR,電気泳動によってλEG10DNAの導入を調べた。導入が確認できた11例の内,12週齢以上に成育した9例をWistar系ラットと交配させ,得られた125例の産子全てにおいてλEG10DNAの導入を調べたところ,56例の個体で確認できた。しかし,これらの個体は表現型が全てヘテロ型であるため,遺伝的に均一なホモ型ではない。今後は56例のヘテロ型の内in vitroパッケージングによるλEG10DNA回収効率の高い個体を選抜し,さらに戻し交配を行うことでホモ型であるTgラットを作出する。Mutations are implicated in the etiology of cancer and other age-related diseases. Some environmental chemicals may increase the occurrence of mutations in human somatic and/or germ cells. Thus, the evaluation of the potential of chemicals to induce mutations is a particularly important issue in the field of environmental mutagenesis and carchinogenesis. Transgenic mice have been produced by introducing λEG10DNA into mice (gpt delta mice), because they are thought to be useful in effectively evaluating the safety of new chemicals in vivo. However, the number of the mice taken as samples is limited, and it is necessary to produce transgenic rats (Tg rats), which are more reproductive and bigger than mice. This study made an attempt to produce Tg rats, which have the same characteristics of gpt delta mice. Wistar rats were used in this study because they have high reproductive efficiency, and are frequently used worldwide. λEG10DNA composed of the Escherichia gpt gene and red/garn gene of λphage was introduced into fertilized eggs of the rats. The embryos were placed in female rat recipients, which had been induced to be pseudopregnant. The recipients had babies by spontaneous delivery. DNA was extracted from the tails of the baby rats to ascertain whether λEG10DNA was introduced in the rats using PCR, and electrophoresis. The introduction was confirmed in eleven baby rats. After that, nine rats out of the eleven, which reached twelve weeks old, were mated with Wistar rats. One hundred twenty-five babies were obtained, and introduction of λEG10DNA was ascertained in fifty-six individuals among the babies. However, all of the fifty-six individuals were of the Hetero phenotype. That is, they were not of the Homo phenotype, which means genetically uniform. We are planning to select individuals with high recovery efficiency of λEG10DNA from the fifty-six individuals by in vitro packaging, and then backcross them in order to produce Homo phenotype Tg rats

トランスジェニックマウスを用いたコプラナーPCBsの毒性評価 : 化学物質の安全性評価モデルTgラットの開発

突然変異は,癌や加齢にともなう疾患などの原因と考えられているし,いくつかの環境中の化学物質は,人の体細胞や生殖細胞における突然変異の発生を増加させているとも考えられている。そのため,突然変異を誘発するような化学物質の安全性を評価することは,変異原誘発性や発癌性のフィールドでは特に重要な問題となっている。このことから遺伝子突然変異の有無を効率良く評価できる有用なモデルとしてλEG10DNAを導入したTgマウス(gpt delta)が作出され,生体に対する化学物質の安全性を評価に用いられている。しかし,マウスでは採取できるサンプル量などに限りがあるため,マウスよりも大きいラットでのTg個体の作出が切望されている。本研究はgpt deltaマウスと同じ特性を持つTgラットの作出を試みた。ラットは繁殖効率が高く,世界的に使用頻度が高いWistar系ラットを用いた。大腸菌gpt遺伝子とλファージのred/gam遺伝子から構成されるλEG10DNAをラット受精卵に導入した。これらの胚を偽妊娠誘起したレシピエント雌へ移植し,自然分娩によって産子を得た。得られた産子の尾よりDNAを抽出し,PCR,電気泳動によってλEG10DNAの導入を調べた。導入が確認できた11例の内,12週齢以上に成育した9例をWistar系ラットと交配させ,得られた125例の産子全てにおいてλEG10DNAの導入を調べたところ,56例の個体で確認できた。しかし,これらの個体は表現型が全てヘテロ型であるため,遺伝的に均一なホモ型ではない。今後は56例のヘテロ型の内in vitroパッケージングによるλEG10DNA回収効率の高い個体を選抜し,さらに戻し交配を行うことでホモ型であるTgラットを作出する。Mutations are implicated in the etiology of cancer and other age-related diseases. Some environmental chemicals may increase the occurrence of mutations in human somatic and/or germ cells. Thus, the evaluation of the potential of chemicals to induce mutations is a particularly important issue in the field of environmental mutagenesis and carchinogenesis. Transgenic mice have been produced by introducing λEG10DNA into mice (gpt delta mice), because they are thought to be useful in effectively evaluating the safety of new chemicals in vivo. However, the number of the mice taken as samples is limited, and it is necessary to produce transgenic rats (Tg rats), which are more reproductive and bigger than mice. This study made an attempt to produce Tg rats, which have the same characteristics of gpt delta mice. Wistar rats were used in this study because they have high reproductive efficiency, and are frequently used worldwide. λEG10DNA composed of the Escherichia gpt gene and red/garn gene of λphage was introduced into fertilized eggs of the rats. The embryos were placed in female rat recipients, which had been induced to be pseudopregnant. The recipients had babies by spontaneous delivery. DNA was extracted from the tails of the baby rats to ascertain whether λEG10DNA was introduced in the rats using PCR, and electrophoresis. The introduction was confirmed in eleven baby rats. After that, nine rats out of the eleven, which reached twelve weeks old, were mated with Wistar rats. One hundred twenty-five babies were obtained, and introduction of λEG10DNA was ascertained in fifty-six individuals among the babies. However, all of the fifty-six individuals were of the Hetero phenotype. That is, they were not of the Homo phenotype, which means genetically uniform. We are planning to select individuals with high recovery efficiency of λEG10DNA from the fifty-six individuals by in vitro packaging, and then backcross them in order to produce Homo phenotype Tg rats

トランスジェニックマウスを用いたコプラナーPCBsの毒性評価 : 化学物質の安全性評価モデルTgラットの開発

突然変異は,癌や加齢にともなう疾患などの原因と考えられているし,いくつかの環境中の化学物質は,人の体細胞や生殖細胞における突然変異の発生を増加させているとも考えられている。そのため,突然変異を誘発するような化学物質の安全性を評価することは,変異原誘発性や発癌性のフィールドでは特に重要な問題となっている。このことから遺伝子突然変異の有無を効率良く評価できる有用なモデルとしてλEG10DNAを導入したTgマウス(gpt delta)が作出され,生体に対する化学物質の安全性を評価に用いられている。しかし,マウスでは採取できるサンプル量などに限りがあるため,マウスよりも大きいラットでのTg個体の作出が切望されている。本研究はgpt deltaマウスと同じ特性を持つTgラットの作出を試みた。ラットは繁殖効率が高く,世界的に使用頻度が高いWistar系ラットを用いた。大腸菌gpt遺伝子とλファージのred/gam遺伝子から構成されるλEG10DNAをラット受精卵に導入した。これらの胚を偽妊娠誘起したレシピエント雌へ移植し,自然分娩によって産子を得た。得られた産子の尾よりDNAを抽出し,PCR,電気泳動によってλEG10DNAの導入を調べた。導入が確認できた11例の内,12週齢以上に成育した9例をWistar系ラットと交配させ,得られた125例の産子全てにおいてλEG10DNAの導入を調べたところ,56例の個体で確認できた。しかし,これらの個体は表現型が全てヘテロ型であるため,遺伝的に均一なホモ型ではない。今後は56例のヘテロ型の内in vitroパッケージングによるλEG10DNA回収効率の高い個体を選抜し,さらに戻し交配を行うことでホモ型であるTgラットを作出する。Mutations are implicated in the etiology of cancer and other age-related diseases. Some environmental chemicals may increase the occurrence of mutations in human somatic and/or germ cells. Thus, the evaluation of the potential of chemicals to induce mutations is a particularly important issue in the field of environmental mutagenesis and carchinogenesis. Transgenic mice have been produced by introducing λEG10DNA into mice (gpt delta mice), because they are thought to be useful in effectively evaluating the safety of new chemicals in vivo. However, the number of the mice taken as samples is limited, and it is necessary to produce transgenic rats (Tg rats), which are more reproductive and bigger than mice. This study made an attempt to produce Tg rats, which have the same characteristics of gpt delta mice. Wistar rats were used in this study because they have high reproductive efficiency, and are frequently used worldwide. λEG10DNA composed of the Escherichia gpt gene and red/garn gene of λphage was introduced into fertilized eggs of the rats. The embryos were placed in female rat recipients, which had been induced to be pseudopregnant. The recipients had babies by spontaneous delivery. DNA was extracted from the tails of the baby rats to ascertain whether λEG10DNA was introduced in the rats using PCR, and electrophoresis. The introduction was confirmed in eleven baby rats. After that, nine rats out of the eleven, which reached twelve weeks old, were mated with Wistar rats. One hundred twenty-five babies were obtained, and introduction of λEG10DNA was ascertained in fifty-six individuals among the babies. However, all of the fifty-six individuals were of the Hetero phenotype. That is, they were not of the Homo phenotype, which means genetically uniform. We are planning to select individuals with high recovery efficiency of λEG10DNA from the fifty-six individuals by in vitro packaging, and then backcross them in order to produce Homo phenotype Tg rats