In Vivo Bac-Based Gene Delivery Towards Murine Pancreatic Parenchyma Confers Sustained Expression of Gene of Interest

Overview

Journal Int J Mol Sci

Publisher MDPI

Specialties Biochemistry
Chemistry
Molecular Biology

Date 2019 Jun 29

PMID 31247905

Citations 1

Authors

Masahiro Sato

Emi Inada

Issei Saitoh

Shingo Nakamura

Satoshi Watanabe

Affiliations

Soon will be listed here.

Abstract

The pancreas is a glandular organ that functions in the digestive system and endocrine system of vertebrates. The most common disorders involving the pancreas are diabetes, pancreatitis, and pancreatic cancer. In vivo gene delivery targeting the pancreas is important for preventing or curing such diseases and for exploring the biological function of genes involved in the pathogenesis of these diseases. Our previous experiments demonstrated that adult murine pancreatic cells can be efficiently transfected by exogenous plasmid DNA following intraparenchymal injection and subsequent in vivo electroporation using tweezer-type electrodes. Unfortunately, the induced gene expression was transient. Transposon-based gene delivery, such as that facilitated by Bac (PB), is known to confer stable integration of a gene of interest (GOI) into host chromosomes, resulting in sustained expression of the GOI. In this study, we investigated the use of the PB transposon system to achieve stable gene expression when transferred into murine pancreatic cells using the above-mentioned technique. Expression of the GOI (coding for fluorescent protein) continued for at least 1.5 months post-gene delivery. Splinkerette-PCR-based analysis revealed the presence of the consensus sequence TTAA at the junctional portion between host chromosomes and the transgenes; however, this was not observed in all samples. This plasmid-based PB transposon system enables constitutive expression of the GOI in pancreas for potential therapeutic and biological applications.

Citing Articles

Bac-Based Non-Viral In Vivo Gene Delivery Useful for Production of Genetically Modified Animals and Organs.

Sato M, Inada E, Saitoh I, Watanabe S, Nakamura S Pharmaceutics. 2020; 12(3).

PMID: 32204422 PMC: 7151002. DOI: 10.3390/pharmaceutics12030277.

References

Bauser C, Elick T, Fraser M . Proteins from nuclear extracts of two lepidopteran cell lines recognize the ends of TTAA-specific transposons piggyBac and tagalong. Insect Mol Biol. 1999; 8(2):223-30. DOI: 10.1046/j.1365-2583.1999.820223.x. View

Yant S, Meuse L, Chiu W, Ivics Z, Izsvak Z, Kay M . Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat Genet. 2000; 25(1):35-41. DOI: 10.1038/75568. View

Somiari S, Drabick J, GILBERT R, Heller R, Jaroszeski M, Malone R . Theory and in vivo application of electroporative gene delivery. Mol Ther. 2000; 2(3):178-87. DOI: 10.1006/mthe.2000.0124. View

Shifrin A, Auricchio A, Yu Q, Wilson J, Raper S . Adenoviral vector-mediated insulin gene transfer in the mouse pancreas corrects streptozotocin-induced hyperglycemia. Gene Ther. 2001; 8(19):1480-9. DOI: 10.1038/sj.gt.3301544. View

Wang A, Peng P, Ehrhardt A, Storm T, Kay M . Comparison of adenoviral and adeno-associated viral vectors for pancreatic gene delivery in vivo. Hum Gene Ther. 2004; 15(4):405-13. DOI: 10.1089/104303404322959551. View

Yu L, Lin B, Zhang Z, Guo L . Direct transfer of A20 gene into pancreas protected mice from streptozotocin-induced diabetes. Acta Pharmacol Sin. 2004; 25(6):721-6. View

Ayuso E, Chillon M, Agudo J, Haurigot V, Bosch A, Carretero A . In vivo gene transfer to pancreatic beta cells by systemic delivery of adenoviral vectors. Hum Gene Ther. 2004; 15(8):805-12. DOI: 10.1089/1043034041648426. View

Ding S, Wu X, Li G, Han M, Zhuang Y, Xu T . Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell. 2005; 122(3):473-83. DOI: 10.1016/j.cell.2005.07.013. View

Ayuso E, Chillon M, Garcia F, Agudo J, Andaluz A, Carretero A . In vivo gene transfer to healthy and diabetic canine pancreas. Mol Ther. 2005; 13(4):747-55. DOI: 10.1016/j.ymthe.2005.10.017. View

10.

Wang Z, Zhu T, Rehman K, Bertera S, Zhang J, Chen C . Widespread and stable pancreatic gene transfer by adeno-associated virus vectors via different routes. Diabetes. 2006; 55(4):875-84. DOI: 10.2337/diabetes.55.04.06.db05-0927. View

11.

Niwa H, Yamamura K, Miyazaki J . Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991; 108(2):193-9. DOI: 10.1016/0378-1119(91)90434-d. View

12.

Wu S, James Meir Y, Coates C, Handler A, Pelczar P, Moisyadi S . piggyBac is a flexible and highly active transposon as compared to sleeping beauty, Tol2, and Mos1 in mammalian cells. Proc Natl Acad Sci U S A. 2006; 103(41):15008-13. PMC: 1622771. DOI: 10.1073/pnas.0606979103. View

13.

Wilson M, Coates C, George Jr A . PiggyBac transposon-mediated gene transfer in human cells. Mol Ther. 2006; 15(1):139-45. DOI: 10.1038/sj.mt.6300028. View

14.

Clark K, Carlson D, Foster L, Kong B, Foster D, Fahrenkrug S . Enzymatic engineering of the porcine genome with transposons and recombinases. BMC Biotechnol. 2007; 7:42. PMC: 1939997. DOI: 10.1186/1472-6750-7-42. View

15.

Wang W, Lin C, Lu D, Ning Z, Cox T, Melvin D . Chromosomal transposition of PiggyBac in mouse embryonic stem cells. Proc Natl Acad Sci U S A. 2008; 105(27):9290-5. PMC: 2440425. DOI: 10.1073/pnas.0801017105. View

16.

Keith J, Schaeper C, Fraser T, Fraser Jr M . Mutational analysis of highly conserved aspartate residues essential to the catalytic core of the piggyBac transposase. BMC Mol Biol. 2008; 9:73. PMC: 2533014. DOI: 10.1186/1471-2199-9-73. View

17.

Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjen K . Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature. 2009; 458(7239):771-5. PMC: 2667910. DOI: 10.1038/nature07864. View

18.

Woltjen K, Michael I, Mohseni P, Desai R, Mileikovsky M, Hamalainen R . piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature. 2009; 458(7239):766-70. PMC: 3758996. DOI: 10.1038/nature07863. View

19.

Yusa K, Rad R, Takeda J, Bradley A . Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat Methods. 2009; 6(5):363-9. PMC: 2677165. DOI: 10.1038/nmeth.1323. View

20.

Ivics Z, Li M, Mates L, Boeke J, Nagy A, Bradley A . Transposon-mediated genome manipulation in vertebrates. Nat Methods. 2009; 6(6):415-22. PMC: 2867038. DOI: 10.1038/nmeth.1332. View