- Email: [email protected]
Construction and Characterization of a cDNA Library from Liver Tissue of Chinese Banna Minipig Inbred Line
Construction and Characterization of a cDNA Library from Liver Tissue of Chinese Banna Minipig Inbred Line W. Tan, Y. Chen, L. Zhang, Y. Lu, S. Li, R. Zeng, Y. Zeng, Y. Li, and J. Cheng ABSTRACT A xenograft that performs efficient functions is an essential premise for successful xenotransplantation. Our early study indicated that Chinese Banna minipig inbred line (BMI) was an ideal xenograft donor. However, the activities of some proteins synthesized by the BMI liver are different from the human, which could lead to functional disorders in coagulation, fibrinolysis, and anticoagulation after liver xenotransplantation. Therefore, it is important to investigate the genetic background of protein incompatibility and to provide new strategies for gene manipulation. In this study we constructed a cDNA expression library using BMI liver tissue to obtain an understanding of nucleic acid and protein differences between the two species. We extracted total RNA and purified mRNA of the liver tissue from one of the sixteenth inbred generation of BMI/JS 151 substrain. After double-strand cDNA synthesis, we fractionated it on a CHROMA APIN-400 column; ligated the longer than 500bp cDNA into a ZAP Express Vector; and performed a : phage packaging reaction, library amplification, and titer. We randomly picked 12 plaques and tested the length of inserts. The titers of the primary and amplified libraries were 1.0 ⫻ 106 pfu/mL and 5.0 ⫻ 109 pfu/mL, respectively. The percentages of recombinants were 97.0% in the primary library and 98.0% in the amplified library. The lengths of most inserts were between 750 bp and 2.0 kb. Thus, we successfully constructed a cDNA expression library from BMI liver tissue. Using the library, we hope to get a full-length cDNA of some important genes and conduct further studies on porcine liver function in xenotransplantation.
P
IG-TO-HUMAN xenotransplantation is considered a possible solution to the discrepancy between the demand and supply of organs for clinical transplantation.1 The liver is a multifunction organ that is involved in a number of excretory, synthetic, and metabolic functions, including the synthesis of most plasma proteins and main detoxification of exogenous compounds such as drugs and toxins.2 So hepatic function matching is an important aspect of successful xenotransplantation. The Banna minipig inbred line (BMI) is a unique inbred pig. Because of their clear genetic background and minor interindividual differences, BMI are suitable for pig-to-human xenotransplantation. In our previous study, we compared the synthesis, metabolism, and drainage functions of the liver from BMI versus humans. The results showed a few differences that we did not regard as significant.3–5 However, their molecular compatibilities and regulatory mechanisms are still unclear. A cDNA library containing the information encoded by the messenger RNA(mRNA) of a particular tissue or organism is
useful tool for research on gene structure, function, and manipulation.6 In this study, we constructed a liver tissue cDNA BMI library to discover and investigate relevant genes in xenotransplantation.
From the Key Laboratory of Transplant Engineering and Immunology (W.T., Y.C., L.Z., Y.Lu., S.L., Y.Li., J.C.), Ministry of Health, West China Hospital, Sichuan University, Chengdu; and Lab of Banna Minipig Inbred Line, Yunnan Agricultural University (R.Z., Y.Z.), Kunming, P.R. China. Supported by National Basic Research Program of China, No. 2003CB515504; National Basic Research Program of China, No. 2004CCA01800; and Program for Changjiang Scholars and Innovative Research Team in University, Ministry of Education. Address reprint requests to Jing Qiu Cheng, PhD, Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu 610041, P.R. China. E-mail: [email protected]
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.06.095
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2264
Transplantation Proceedings, 38, 2264 –2266 (2006)
TISSUE OF CHINESE BANNA MINIPIG
2265
MATERIALS AND METHODS Subjects The liver tissue was sampled from a healthy 6-year-old male pig from the sixteenth inbred generation of the BMI/JS 151 substrain, which was bought from Yunnan Agricultural University, with consent to excise a part of the liver. The excised liver tissue was placed into liquid nitrogen immediately.
Reagents and Apparatus Trizol reagent was bought from Invitrogen Co(Lot no. 1232134); Oligotex mRNA Mini Kit, from Qiagen Co (Lot no. 11879749); ZAP Express cDNA Synthesis Kit and ZAP Express cDNA Gigapack III Gold Cloning Kit, from Stratagene Co (Lot no. 0540377); and CHROMA SPIN⫹TE-400, from BD Co (Lot no. 5050459). We used Bio-Rad PowerPac-300 Power Supply, Bio-Rad minisubcellGT Electrophoresis Cell, Bio-Rad Universal Hood II Gel Documentation Family and Bio-Rad SmartSpec Plus Spectrophotometer for assays.
Purification and Quantification of RNA Total RNA from the BMI liver was isolated using Trizol reagents, followed by Poly (A)⫹ RNA purification using Oligotex polystyrene-latex particles. RNA was quantified by measuring the optical density of a dilute RNA solution. The integrity of the RNA was analyzed using 1.1% agarose/GoldView gel electrophoresis with RNA markers. The purity of the RNA was checked by the ratio of OD260/OD280.
Construction of the cDNA Library Five micrograms of the BMI liver Poly (A)⫹ RNA was used to synthesize cDNA using the ZAP Express cDNA Synthesis Kit. Five microliters of the first-strand cDNA and 1 g of the doublestranded cDNA were taken for analysis by electrophoresis in 1.1% agarose/GoldView gel. Then EcoR I adapters were ligated to the blunted cDNA. After Xho I digestion, cDNA size fractionation was performed using CHROMA SPIN-400 columns. cDNA fractions longer than 500 bp were precipitated by ethanol and glycogen. After being resolved and accurately quantified, cDNA was ligated with the ZAP Express vector. The lambda packaging reaction to produce the primary cDNA library in vitro was established according to the manufacturer’s instructions of ZAP Express cDNA Gigapack III Gold extract. To make a large, stable quantity of a high-titer stock of the library, we amplified the primary cDNA library.
Identification of the cDNA Library According to the protocol, the unamplified and amplified cDNA libraries were titered and the percentage of recombinant clones determined by blue and white screen. To identify the cDNA inserts of the recombinants, 12 plaques were randomly picked from plate. Then PCR was performed with T3, T7 general primers and the products checked on a 1.1% agarose gel.
RESULTS
In our study, we obtained 1.98 mg total RNA and 10.65 g Poly (A)⫹ RNA from 1.2 g BMI liver tissue. The total RNA appeared as clear bands of 28S and 18S, and the Poly (A)⫹ RNA, as a long smear from 500 bp to 6 kb or longer (Fig 1).
Fig 1. 1.1% agarose gel electrophoresis of total RNA and Poly(A)⫹ RNA. 1. RNA Marker RL6000; 2. total RNA; 3. Poly (A)⫹ RNA.
The ratio of OD260/280 to the total RNA and Poly (A)⫹ RNA was 2.1. Both the long smear in first-strand cDNA and the double-stranded cDNA distributed from 250 bp to 6 kb or longer (Fig 2), which indicated that double-strandedcDNA was successfully synthesized. The four fractions containing cDNA were longer than 500 bp (Fig 3). The titers of primary and amplified libraries were 1.0 ⫻ 106 pfu/mL and 5.0 ⫻ 109 pfu/mL, respectively. The percentages of recombinants were 97.0% in primary library and 98.0% in amplified library. The sizes of most inserts among the PCR products were between 750 to 2000 bp with some larger than 2 kb (Fig 4). DISCUSSION
A cDNA library is a useful tool for functional genomics. In this study, a BMI liver tissue cDNA library was constructed with the ZAP Express vector. This library of both eukaryotic and prokaryotic expression may offer the opportunity to identify and characterize relevant genes of the BMI genome. According to Clareke-Carbon’s formula, a cDNA library should theoretically contain at least 3.3 ⫻ 105 independent clones so that a clone derived from a lowabundance mRNA would be screened out with 99 percent probability from the library.7 A constructed primary cDNA library could be screened directly. However, library amplification is necessary because of the limited volume and insufficient stability of the primary library; 1 ⫻ 109 pfu/mL is a desirable titer for the amplified library. The capacities of the primary and amplified cDNA library that we constructed were 1 ⫻ 106 pfu/mL and 5 ⫻ 109 pfu/mL respectively, which should meet almost all requirements to find a cDNA clone derived from a low-abundance mRNA. The average full length of mRNA translated by most
2266
Fig 2. 1.1% agarose gel electrophoresis of the first-strand cDNA and the double-stranded cDNA. 1. DNA Marker DL2000; 2. -Hind III digest DNA Marker; 3. the first-strand cDNA; 4. the double-stranded cDNA.
functional genes of human genome is 2000 bp, so a useful cDNA library should have clones whose inserts are beyond 1200 bp.7 Our results showed that the library we constructed has inserts between 500 and 2000 bp or longer, most beyond 1200 bp. Thus it reaches the requirement for further studies on gene structure, translation, and expression. Our previous study showed that the BMI coagulation factors XII, VII, and X triggered human intrinsic, extrinsic, and common clotting pathways but probably had stronger activities.5 So the transplanted porcine graft may produce functional coagulation factors leading human blood to clot normally or more quickly. On the other hand, BMI liver showed a stronger capability for bilirubin elimination, enzyme activity, and drug metabolism. These results suggested that coagulation, anticoagulation, and metabolic disorders may occur after transplanting this porcine liver into the human body. Due to the development of bioinformatics, the sequence, and structure of proteins may predicted and their functions speculated by gene sequence analysis. Furthermore, comparing homologous gene sequence from various species may indicate differences in protein function. Much evidence has supported the fact that as one of the large
Fig 3. 1.1% agarose gel electrophoresis of the fractionated cDNA. 1– 4 and 6 – 8 fractionated cDNA; 5 DNA Marker DL2000.
TAN, CHEN, ZHANG ET AL
Fig 4. 1.1% agarose gel electrophoresis of PCR products of inserts. 1. DNA Marker DL2000; 2–13. Products of inserts.
mammals, the pig is highly similar to the human in genetic background. The homology of many known genes between human and pig was higher than many other mammalians, such as rat, sheep, or cow. However, due to the unfinished pig genome project, our knowledge of pig gene structure and expression remains rudimentary level. In this study we have described a liver tissue cDNA expression library of Chinese Bana Minipig Inbred Line, a unique inbred line of minipig. By analyzing homology of hepatic proteins and differences in nucleotide, amino acid sequences, and motif structures between BMI and humans, we hope to obtain useful information about the function of porcine proteins in the human body. We hope to provide significant genetic evidence on the unclear question of whether a BMI liver graft could function in the human body. Furthermore, the rich genetic information of the library may assist further research on the immunology, physiology, and pharmacogenetics of xenotransplantation, providing useful clues and theoretical support to the manipulation of potential gene targets. REFERENCES 1. Chen D, Morgan F, Berton I, et al: Developing a porcine transplantation model: efficient gene transfer into porcine vascular cells. Transplantation 77:1443, 2004 2. Corrigan JJ, Jeter M, Earnest DL: Prothrombin antigen and coagulant activity in patients with liver disease. JAMA 248:1736, 1982 3. Zhang L, Sun X, Cheng J, et al: Study of hepatic function matching between Banna Minipig inbred and humans. Transplant Proc 36:2492, 2004 4. Zhang L, Li Y, Liu J, et al: Activation of human coagulation system by liver-derived clotting factors of Banna Minipig inbred line. Transplant Proc 36:2490, 2004 5. Zhang L, Li Y, Jiang H, et al: Comparison of hepatic coagulant, fibrinolytic, and anticoagulant functions between Banna Minipig inbred line and humans. Transplantation 79:1, 2005 6. Ying S: Complementary DNA libraries. Mol Biotech 27:245, 2004 7. Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Lab Press; 2001, p 11
P
IG-TO-HUMAN xenotransplantation is considered a possible solution to the discrepancy between the demand and supply of organs for clinical transplantation.1 The liver is a multifunction organ that is involved in a number of excretory, synthetic, and metabolic functions, including the synthesis of most plasma proteins and main detoxification of exogenous compounds such as drugs and toxins.2 So hepatic function matching is an important aspect of successful xenotransplantation. The Banna minipig inbred line (BMI) is a unique inbred pig. Because of their clear genetic background and minor interindividual differences, BMI are suitable for pig-to-human xenotransplantation. In our previous study, we compared the synthesis, metabolism, and drainage functions of the liver from BMI versus humans. The results showed a few differences that we did not regard as significant.3–5 However, their molecular compatibilities and regulatory mechanisms are still unclear. A cDNA library containing the information encoded by the messenger RNA(mRNA) of a particular tissue or organism is
useful tool for research on gene structure, function, and manipulation.6 In this study, we constructed a liver tissue cDNA BMI library to discover and investigate relevant genes in xenotransplantation.
From the Key Laboratory of Transplant Engineering and Immunology (W.T., Y.C., L.Z., Y.Lu., S.L., Y.Li., J.C.), Ministry of Health, West China Hospital, Sichuan University, Chengdu; and Lab of Banna Minipig Inbred Line, Yunnan Agricultural University (R.Z., Y.Z.), Kunming, P.R. China. Supported by National Basic Research Program of China, No. 2003CB515504; National Basic Research Program of China, No. 2004CCA01800; and Program for Changjiang Scholars and Innovative Research Team in University, Ministry of Education. Address reprint requests to Jing Qiu Cheng, PhD, Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu 610041, P.R. China. E-mail: [email protected]
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.06.095
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2264
Transplantation Proceedings, 38, 2264 –2266 (2006)
TISSUE OF CHINESE BANNA MINIPIG
2265
MATERIALS AND METHODS Subjects The liver tissue was sampled from a healthy 6-year-old male pig from the sixteenth inbred generation of the BMI/JS 151 substrain, which was bought from Yunnan Agricultural University, with consent to excise a part of the liver. The excised liver tissue was placed into liquid nitrogen immediately.
Reagents and Apparatus Trizol reagent was bought from Invitrogen Co(Lot no. 1232134); Oligotex mRNA Mini Kit, from Qiagen Co (Lot no. 11879749); ZAP Express cDNA Synthesis Kit and ZAP Express cDNA Gigapack III Gold Cloning Kit, from Stratagene Co (Lot no. 0540377); and CHROMA SPIN⫹TE-400, from BD Co (Lot no. 5050459). We used Bio-Rad PowerPac-300 Power Supply, Bio-Rad minisubcellGT Electrophoresis Cell, Bio-Rad Universal Hood II Gel Documentation Family and Bio-Rad SmartSpec Plus Spectrophotometer for assays.
Purification and Quantification of RNA Total RNA from the BMI liver was isolated using Trizol reagents, followed by Poly (A)⫹ RNA purification using Oligotex polystyrene-latex particles. RNA was quantified by measuring the optical density of a dilute RNA solution. The integrity of the RNA was analyzed using 1.1% agarose/GoldView gel electrophoresis with RNA markers. The purity of the RNA was checked by the ratio of OD260/OD280.
Construction of the cDNA Library Five micrograms of the BMI liver Poly (A)⫹ RNA was used to synthesize cDNA using the ZAP Express cDNA Synthesis Kit. Five microliters of the first-strand cDNA and 1 g of the doublestranded cDNA were taken for analysis by electrophoresis in 1.1% agarose/GoldView gel. Then EcoR I adapters were ligated to the blunted cDNA. After Xho I digestion, cDNA size fractionation was performed using CHROMA SPIN-400 columns. cDNA fractions longer than 500 bp were precipitated by ethanol and glycogen. After being resolved and accurately quantified, cDNA was ligated with the ZAP Express vector. The lambda packaging reaction to produce the primary cDNA library in vitro was established according to the manufacturer’s instructions of ZAP Express cDNA Gigapack III Gold extract. To make a large, stable quantity of a high-titer stock of the library, we amplified the primary cDNA library.
Identification of the cDNA Library According to the protocol, the unamplified and amplified cDNA libraries were titered and the percentage of recombinant clones determined by blue and white screen. To identify the cDNA inserts of the recombinants, 12 plaques were randomly picked from plate. Then PCR was performed with T3, T7 general primers and the products checked on a 1.1% agarose gel.
RESULTS
In our study, we obtained 1.98 mg total RNA and 10.65 g Poly (A)⫹ RNA from 1.2 g BMI liver tissue. The total RNA appeared as clear bands of 28S and 18S, and the Poly (A)⫹ RNA, as a long smear from 500 bp to 6 kb or longer (Fig 1).
Fig 1. 1.1% agarose gel electrophoresis of total RNA and Poly(A)⫹ RNA. 1. RNA Marker RL6000; 2. total RNA; 3. Poly (A)⫹ RNA.
The ratio of OD260/280 to the total RNA and Poly (A)⫹ RNA was 2.1. Both the long smear in first-strand cDNA and the double-stranded cDNA distributed from 250 bp to 6 kb or longer (Fig 2), which indicated that double-strandedcDNA was successfully synthesized. The four fractions containing cDNA were longer than 500 bp (Fig 3). The titers of primary and amplified libraries were 1.0 ⫻ 106 pfu/mL and 5.0 ⫻ 109 pfu/mL, respectively. The percentages of recombinants were 97.0% in primary library and 98.0% in amplified library. The sizes of most inserts among the PCR products were between 750 to 2000 bp with some larger than 2 kb (Fig 4). DISCUSSION
A cDNA library is a useful tool for functional genomics. In this study, a BMI liver tissue cDNA library was constructed with the ZAP Express vector. This library of both eukaryotic and prokaryotic expression may offer the opportunity to identify and characterize relevant genes of the BMI genome. According to Clareke-Carbon’s formula, a cDNA library should theoretically contain at least 3.3 ⫻ 105 independent clones so that a clone derived from a lowabundance mRNA would be screened out with 99 percent probability from the library.7 A constructed primary cDNA library could be screened directly. However, library amplification is necessary because of the limited volume and insufficient stability of the primary library; 1 ⫻ 109 pfu/mL is a desirable titer for the amplified library. The capacities of the primary and amplified cDNA library that we constructed were 1 ⫻ 106 pfu/mL and 5 ⫻ 109 pfu/mL respectively, which should meet almost all requirements to find a cDNA clone derived from a low-abundance mRNA. The average full length of mRNA translated by most
2266
Fig 2. 1.1% agarose gel electrophoresis of the first-strand cDNA and the double-stranded cDNA. 1. DNA Marker DL2000; 2. -Hind III digest DNA Marker; 3. the first-strand cDNA; 4. the double-stranded cDNA.
functional genes of human genome is 2000 bp, so a useful cDNA library should have clones whose inserts are beyond 1200 bp.7 Our results showed that the library we constructed has inserts between 500 and 2000 bp or longer, most beyond 1200 bp. Thus it reaches the requirement for further studies on gene structure, translation, and expression. Our previous study showed that the BMI coagulation factors XII, VII, and X triggered human intrinsic, extrinsic, and common clotting pathways but probably had stronger activities.5 So the transplanted porcine graft may produce functional coagulation factors leading human blood to clot normally or more quickly. On the other hand, BMI liver showed a stronger capability for bilirubin elimination, enzyme activity, and drug metabolism. These results suggested that coagulation, anticoagulation, and metabolic disorders may occur after transplanting this porcine liver into the human body. Due to the development of bioinformatics, the sequence, and structure of proteins may predicted and their functions speculated by gene sequence analysis. Furthermore, comparing homologous gene sequence from various species may indicate differences in protein function. Much evidence has supported the fact that as one of the large
Fig 3. 1.1% agarose gel electrophoresis of the fractionated cDNA. 1– 4 and 6 – 8 fractionated cDNA; 5 DNA Marker DL2000.
TAN, CHEN, ZHANG ET AL
Fig 4. 1.1% agarose gel electrophoresis of PCR products of inserts. 1. DNA Marker DL2000; 2–13. Products of inserts.
mammals, the pig is highly similar to the human in genetic background. The homology of many known genes between human and pig was higher than many other mammalians, such as rat, sheep, or cow. However, due to the unfinished pig genome project, our knowledge of pig gene structure and expression remains rudimentary level. In this study we have described a liver tissue cDNA expression library of Chinese Bana Minipig Inbred Line, a unique inbred line of minipig. By analyzing homology of hepatic proteins and differences in nucleotide, amino acid sequences, and motif structures between BMI and humans, we hope to obtain useful information about the function of porcine proteins in the human body. We hope to provide significant genetic evidence on the unclear question of whether a BMI liver graft could function in the human body. Furthermore, the rich genetic information of the library may assist further research on the immunology, physiology, and pharmacogenetics of xenotransplantation, providing useful clues and theoretical support to the manipulation of potential gene targets. REFERENCES 1. Chen D, Morgan F, Berton I, et al: Developing a porcine transplantation model: efficient gene transfer into porcine vascular cells. Transplantation 77:1443, 2004 2. Corrigan JJ, Jeter M, Earnest DL: Prothrombin antigen and coagulant activity in patients with liver disease. JAMA 248:1736, 1982 3. Zhang L, Sun X, Cheng J, et al: Study of hepatic function matching between Banna Minipig inbred and humans. Transplant Proc 36:2492, 2004 4. Zhang L, Li Y, Liu J, et al: Activation of human coagulation system by liver-derived clotting factors of Banna Minipig inbred line. Transplant Proc 36:2490, 2004 5. Zhang L, Li Y, Jiang H, et al: Comparison of hepatic coagulant, fibrinolytic, and anticoagulant functions between Banna Minipig inbred line and humans. Transplantation 79:1, 2005 6. Ying S: Complementary DNA libraries. Mol Biotech 27:245, 2004 7. Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Lab Press; 2001, p 11