- Email: [email protected]
SUMO-1 Genes That Relate to the Recombination Machinery
Isolation and Sequencing of Pig Blm and Ubl-1/SUMO-1 Genes That Relate to the Recombination Machinery K. Matsunami, H. Otsuka, H. Xu, S. Furidaus, A. Ishimaru, M. Fukuzawa, and S. Miyagawa ABSTRACT Background. The gene knockout technique is important in xenotransplantation research. Herein we have described the molecular cloning of two genes that are candidates to overcome the poor rate of homologous recombination. Methods. Candidate cDNA fragments were amplified by polymerase chain reaction (PCR) with the corresponding primer sets deduced from a multiple alignment analysis of other mammalian genes from a cDNA library prepared from pig spleen tissue. To obtain the full-length cDNA, a 5=- and 3=-RACE PCR experiments was performed. Results. We successfully isolated the cDNA sequences of two pig genes—BLM, a Bloom’s syndrome-related gene, and UBL-1/SUMO-1—which are closely related to homologous recombination events. As a result, we verified the sequences of pig BLM and pig UBL-1/SUMO-1. The nucleic acid and amino acid coding sequence homologies of pig BLM gene with the corresponding human gene were 87.3% and 82.9%, respectively. The nucleic acid and amino acid coding sequence homologies of the pig UBL-1/SUMO-1 gene with the human gene were 96.4% and 100%, respectively. Conclusion. Current research into homologous recombination provides the possibility for improvement of gene knockout efficiency by regulating the gene expression profiles of recombination-related genes. Transient interference with the expression of pig UBL-1/ SUMO-1 and BLM is expected to improve gene targeting. The results of the present study provided important information to design siRNA knockdown vectors. They were also useful for ex ante evaluation of expression profiles of these genes in primary cultures of somatic cells, which may enhance the production of gene knockout pigs.
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RODUCTION OF HUMAN embryonic stem cells from skin has been reported by two groups. However, creating functional organs, such as kidney, liver, and heart, is quite difficult. In addition, the human immune system might not be able to eliminate transplanted cells if they should become cancerous. Therefore, xenotransplantation is still one of the most important solutions for the worldwide shortage of organs available for transplantation. Genetic manipulations of pig organs, such as by gene knockout and transgene manipulation, are essential techniques for this field. Recently, our group successfully produced a next-generation, gene-modified pig1 (ie, a homozygous gene knockout of the ␣-1,3-GalT gene with insertion of two transgenes, DAF and N-acetylglucosaminyltransferase-III [GnT-III]).2 However, considering further gene modifications in the future, the gene knockout technique must be improved for successful xenotransplantation.
A human ubiquitin-like protein, Ubl-1/SUMO-1, is associated with Rad51, Rad52 proteins that are involved in homologous recombination. The overexpression of human Rad51 and Rad52 in primate cell lines stimulates homologous recombination events severalfold.3 The process of ubiquitylation is known for its role in targeting proteins for degradation by the proteasome. However, recent studies have identified nonproteolytic functions of ubiquitylation From the Division of Organ Transplantation, Department of Molecular therapeutics, Osaka University Graduate School of Medicine, Osaka, Japan. Address reprint requests to Shuji Miyagawa, Division of Organ Transplantation (E9), Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: [email protected]. osaka-u.ac.jp
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.08.022
Transplantation Proceedings, 40, 2773–2775 (2008)
2773
2774
involving monoubiquitylation of key DNA-repair proteins that have regulatory functions in homologous recombination.4 Overexpression of mutant Ubl-1 that cannot be conjugated to its target proteins inhibits homologous recombination in hamster cells.3 In addition, BLM is one of the RecQ family of DNA helicases; BLM-deficient DT40 cells show enhanced rates of gene targeting.3 The results indicated that transient interference with these genes in pig cells is expected to improve the efficiency of gene targeting. In addition, the selection of pig somatic cells by checking the gene expression profiles of these genes is thought to be useful for gene targeting. Therefore, in this study, we sought to isolate pig homologues of Ubl-1/SUMO-1 and BLM genes and clarify the full-length coding sequences. METHODS Total RNA was isolated from fresh pig spleen tissue using the TRIZOL reagent (Invitrogen).5 The OligoTex-dT30 mRNA purification kit (Takara, Japan) was used for mRNA purification. A portion of purified spleen mRNA was reverse transcribed by RevertraAce reverse transcriptase (TOYOBO, Japan) using random oligo primers to yield a cDNA mixture. We then performed a polymerase chain reaction (PCR) experiment to isolate the candidate cDNA clones using pyrobest DNA polymerase (TOYOBO, Japan) with sense primer (5=-AGGCCGCTGCTGTGCGGAGAC-3=) and antisense primer (5=-AAAAGGAGAGGGCAATATGAAGG-3=) for pig Ubl-1/SUMO-1, and sense primer (5=-GAGGATTATGGCTGCTGTTCCTC-3=) and antisense primer (5=-GCATACGAAGGCTTAAGAAACGGTC-3=) for pig BLM. The amplified DNA fragments were subcloned into the EcoRV site of pBluescript II SK(⫺) cloning vector using the TA-cloning method. The nucleotide sequences were determined by the dideoxy chain termination method, using an ABI PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, Calif, USA). Next, we performed the 5=-
Fig 1. cDNA and deduced amino acid sequences of pig Ubl-1/SUMO-1.
MATSUNAMI, OTSUKA, XU ET AL and 3=-RACE PCR according to the standard method to isolate the whole cDNA sequence. Multiple independent isolated clones were then sequenced to confirm sequence data.
RESULTS
The complete cDNA sequences of the pig Ubl-1/SUMO-1 and pig BLM genes were isolated by the PCR method, with corresponding primers designed from homologous genes of other mammals. Then we used the RACE-PCR method. The results showed that pig Ubl-1/SUMO-1 cDNA composed of 820-bp nucleotide sequences encoded 101 aa (Fig 1). The pig BLM was 4281 bp and 1426 aa (Fig 2). The nucleotide homologies of the pig Ubl-1/SUMO-1 and pig BLM genes compared with the corresponding human genes were 87.3% and 96.4%; the corresponding amino acid sequence homologies were 96.4% and 100%, respectively. DISCUSSION
The gene knockout mouse is now produced routinely in many laboratories. However, with pigs, both homologous recombination in primary somatic cells and then nuclear transfer to pig fertilized eggs is needed because of the inavailability of appropriate pig ES cells. We have already produced an ␣-1,3-GalT knockout pig. However, this required much effort, cost, and time. It is clear that further gene modification, by gene transfection and/or knockout of the undesirable genes, is needed for successful xenotransplantation. Thus, improvement of pig gene knockout techniques is becoming an urgent issue. Current research into homologous recombination offers several promising candidate genes; we successfully isolated pig Ubl-1/SUMO-1 and pig BLM.
PIG BLM AND UBL-SUMO-1 GENES
2775
Fig 2. cDNA and deduced amino acid sequences of pig Blm.
Transient interference with these genes is expected to enhance homologous recombination events and/or the ex ante evaluation of expression profiles of these genes in primary somatic cell cultures. Further studies are needed to establish new approaches to enhance homologous recombination events for future production of gene knockout pigs. REFERENCES 1. Takahagi Y, Fujimura T, Miyagawa S, et al: Production of alpha 1,3-galactosyltransferase gene knockout pigs expressing both
human decay-accelerating factor and N-acetylglucosaminyltransferase III. Mol Reprod Dev 71:331, 2005 2. Miyagawa S, Murakami H, Takahagi Y, et al: Remodeling of the major pig xenoantigen by N-acetylglucosaminyltransferase III in transgenic pig. J Biol Chem 276:39310, 2001 3. Vasquez KM, Marburger K, Intody Z, et al: Manipulating the mammalian genome by homologous recombination. Proc Natl Acad Sci U S A 98:8403, 2001 4. Huang TT, D’andrea AD: Regulation of DNA repair by ubiquitylation. Nature Rev 7:323, 2006 5. Matsunami K, Miyagawa S, Nakagawa K, et al: Molecular cloning of pigGnT-I and 1.2: an application to xenotransplantation. Biochem Biophys Res Commun 343:677, 2006
P
RODUCTION OF HUMAN embryonic stem cells from skin has been reported by two groups. However, creating functional organs, such as kidney, liver, and heart, is quite difficult. In addition, the human immune system might not be able to eliminate transplanted cells if they should become cancerous. Therefore, xenotransplantation is still one of the most important solutions for the worldwide shortage of organs available for transplantation. Genetic manipulations of pig organs, such as by gene knockout and transgene manipulation, are essential techniques for this field. Recently, our group successfully produced a next-generation, gene-modified pig1 (ie, a homozygous gene knockout of the ␣-1,3-GalT gene with insertion of two transgenes, DAF and N-acetylglucosaminyltransferase-III [GnT-III]).2 However, considering further gene modifications in the future, the gene knockout technique must be improved for successful xenotransplantation.
A human ubiquitin-like protein, Ubl-1/SUMO-1, is associated with Rad51, Rad52 proteins that are involved in homologous recombination. The overexpression of human Rad51 and Rad52 in primate cell lines stimulates homologous recombination events severalfold.3 The process of ubiquitylation is known for its role in targeting proteins for degradation by the proteasome. However, recent studies have identified nonproteolytic functions of ubiquitylation From the Division of Organ Transplantation, Department of Molecular therapeutics, Osaka University Graduate School of Medicine, Osaka, Japan. Address reprint requests to Shuji Miyagawa, Division of Organ Transplantation (E9), Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: [email protected]. osaka-u.ac.jp
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.08.022
Transplantation Proceedings, 40, 2773–2775 (2008)
2773
2774
involving monoubiquitylation of key DNA-repair proteins that have regulatory functions in homologous recombination.4 Overexpression of mutant Ubl-1 that cannot be conjugated to its target proteins inhibits homologous recombination in hamster cells.3 In addition, BLM is one of the RecQ family of DNA helicases; BLM-deficient DT40 cells show enhanced rates of gene targeting.3 The results indicated that transient interference with these genes in pig cells is expected to improve the efficiency of gene targeting. In addition, the selection of pig somatic cells by checking the gene expression profiles of these genes is thought to be useful for gene targeting. Therefore, in this study, we sought to isolate pig homologues of Ubl-1/SUMO-1 and BLM genes and clarify the full-length coding sequences. METHODS Total RNA was isolated from fresh pig spleen tissue using the TRIZOL reagent (Invitrogen).5 The OligoTex-dT30 mRNA purification kit (Takara, Japan) was used for mRNA purification. A portion of purified spleen mRNA was reverse transcribed by RevertraAce reverse transcriptase (TOYOBO, Japan) using random oligo primers to yield a cDNA mixture. We then performed a polymerase chain reaction (PCR) experiment to isolate the candidate cDNA clones using pyrobest DNA polymerase (TOYOBO, Japan) with sense primer (5=-AGGCCGCTGCTGTGCGGAGAC-3=) and antisense primer (5=-AAAAGGAGAGGGCAATATGAAGG-3=) for pig Ubl-1/SUMO-1, and sense primer (5=-GAGGATTATGGCTGCTGTTCCTC-3=) and antisense primer (5=-GCATACGAAGGCTTAAGAAACGGTC-3=) for pig BLM. The amplified DNA fragments were subcloned into the EcoRV site of pBluescript II SK(⫺) cloning vector using the TA-cloning method. The nucleotide sequences were determined by the dideoxy chain termination method, using an ABI PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, Calif, USA). Next, we performed the 5=-
Fig 1. cDNA and deduced amino acid sequences of pig Ubl-1/SUMO-1.
MATSUNAMI, OTSUKA, XU ET AL and 3=-RACE PCR according to the standard method to isolate the whole cDNA sequence. Multiple independent isolated clones were then sequenced to confirm sequence data.
RESULTS
The complete cDNA sequences of the pig Ubl-1/SUMO-1 and pig BLM genes were isolated by the PCR method, with corresponding primers designed from homologous genes of other mammals. Then we used the RACE-PCR method. The results showed that pig Ubl-1/SUMO-1 cDNA composed of 820-bp nucleotide sequences encoded 101 aa (Fig 1). The pig BLM was 4281 bp and 1426 aa (Fig 2). The nucleotide homologies of the pig Ubl-1/SUMO-1 and pig BLM genes compared with the corresponding human genes were 87.3% and 96.4%; the corresponding amino acid sequence homologies were 96.4% and 100%, respectively. DISCUSSION
The gene knockout mouse is now produced routinely in many laboratories. However, with pigs, both homologous recombination in primary somatic cells and then nuclear transfer to pig fertilized eggs is needed because of the inavailability of appropriate pig ES cells. We have already produced an ␣-1,3-GalT knockout pig. However, this required much effort, cost, and time. It is clear that further gene modification, by gene transfection and/or knockout of the undesirable genes, is needed for successful xenotransplantation. Thus, improvement of pig gene knockout techniques is becoming an urgent issue. Current research into homologous recombination offers several promising candidate genes; we successfully isolated pig Ubl-1/SUMO-1 and pig BLM.
PIG BLM AND UBL-SUMO-1 GENES
2775
Fig 2. cDNA and deduced amino acid sequences of pig Blm.
Transient interference with these genes is expected to enhance homologous recombination events and/or the ex ante evaluation of expression profiles of these genes in primary somatic cell cultures. Further studies are needed to establish new approaches to enhance homologous recombination events for future production of gene knockout pigs. REFERENCES 1. Takahagi Y, Fujimura T, Miyagawa S, et al: Production of alpha 1,3-galactosyltransferase gene knockout pigs expressing both
human decay-accelerating factor and N-acetylglucosaminyltransferase III. Mol Reprod Dev 71:331, 2005 2. Miyagawa S, Murakami H, Takahagi Y, et al: Remodeling of the major pig xenoantigen by N-acetylglucosaminyltransferase III in transgenic pig. J Biol Chem 276:39310, 2001 3. Vasquez KM, Marburger K, Intody Z, et al: Manipulating the mammalian genome by homologous recombination. Proc Natl Acad Sci U S A 98:8403, 2001 4. Huang TT, D’andrea AD: Regulation of DNA repair by ubiquitylation. Nature Rev 7:323, 2006 5. Matsunami K, Miyagawa S, Nakagawa K, et al: Molecular cloning of pigGnT-I and 1.2: an application to xenotransplantation. Biochem Biophys Res Commun 343:677, 2006