RESUMO
Genetically modified pigs that express fluorescent proteins such as green and red fluorescent proteins have become indispensable biomedical research tools in recent years. Cell or tissue transplantation studies using fluorescent markers should be conducted, wherein the xeno-antigenicity of the fluorescent proteins does not affect engraftment or graft survival. Thus, we aimed to create a transgenic (Tg)-cloned pig that was immunologically tolerant to fluorescent protein antigens. In the present study, we generated a Tg-cloned pig harboring a derivative of Plum modified by a single amino acid substitution in the chromophore. The cells and tissues of this Tg-cloned pig expressing the modified Plum (mPlum) did not fluoresce. However, western blot and immunohistochemistry analyses clearly showed that the mPlum had the same antigenicity as Plum. Thus, we have obtained primary proof of principle for creating a cloned pig that is immunologically tolerant to fluorescent protein antigens.
Assuntos
Animais Geneticamente Modificados , Técnicas de Transferência Nuclear , Transgenes , Animais , Antígenos/metabolismo , Núcleo Celular/metabolismo , Clonagem Molecular , Metilação de DNA , Feminino , Fibroblastos/metabolismo , Citometria de Fluxo , Fluorescência , Vetores Genéticos , Genótipo , Sobrevivência de Enxerto , Imuno-Histoquímica , Substâncias Luminescentes/química , SuínosRESUMO
Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.
Assuntos
Animais Geneticamente Modificados/genética , Galactosiltransferases/genética , Técnicas de Inativação de Genes/veterinária , Oxigenases de Função Mista/genética , Sus scrofa/genética , Alelos , Animais , Animais Geneticamente Modificados/metabolismo , Animais Recém-Nascidos , Linhagem Celular , Clonagem de Organismos/veterinária , Transferência Embrionária/veterinária , Éxons , Feminino , Galactosiltransferases/antagonistas & inibidores , Galactosiltransferases/metabolismo , Homozigoto , Japão , Masculino , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/metabolismo , Técnicas de Transferência Nuclear/veterinária , RNA/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Sus scrofa/metabolismoRESUMO
OBJECTIVES: The aim of the study was to accurately examine the functional volume (RI-vol) of the hepatic segments on single photon emission computed tomography/computed tomography (CT) fusion imaging by technetium-99m galactosyl human serum albumin scintigraphy and compare it with the RI-vol and morphological volume obtained on computed tomography (CT-vol). METHODS: In 60 patients with various liver background statuses who had undergone hepatectomy, the RI-vol and CT-vol were examined in each sector using imaging analysis. The values from a control group (n=91) were used as reference data. RESULTS: The mean RI-vol and CT-vol of the right liver were 64 ± 10 and 63 ± 6%, respectively, whereas the values for the left liver were 36 ± 10 and 37 ± 6%, respectively. Compared with the control group, the ratios in each hemiliver were similar. The mean RI-vol and CT-vol for each sector were also similar, and significant positive correlations were identified between the two volumes (P<0.01). In four patients with hepatic tumors involving the main hepatic vessels or the bile duct and in 10 patients who had undergone portal vein embolization, the actual RI-vol in the injured sector was significantly decreased compared with CT-vol (P<0.05). There were marked changes in functional volume in segment 6+7 and segment 2+3 after portal vein embolization (P<0.05). CONCLUSION: Volumetric measurement using single photon emission computed tomography/CT imaging with technetium-99m galactosyl human serum albumin scintigraphy is useful for evaluating the functional volume in separated livers and offers a good reflection of the background liver status.
