Expression Cloning of Recombinant Escherichia coli lacZ Genes Encoding Cytoplasmic and Nuclear P-galactosidase Variants

Document Type: Original Article

Authors

1 Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran

2 Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands

Abstract

Objective(s)
Nonviral vector can be an attractive alternative to gene delivery in experimental study. In spite of some advantages in comparison with the viral vectors, there are still some limitations for efficiency of gene delivery in nonviral vectors. To determine the effective expression, the recombinant Escherichia coli lacZ genes were cloned into the different variants of pcDNA3.1 and then the mammalian cells were transfected.
Methods and Materials
The coding sequences of cytoplasmic and nuclear variants of lacZ gene were inserted downstream of the human cytomegalovirus immediate-early gene promoter of plasmid pcDNA3.1/myc-His C. The new cytoplasmic and nuclear constricts of E. coli p-galactosidase-coding sequences were introduced into HeLa cells with the aid of linear polyethylenimine and at 2 days post-transfection the cells were stained using 5- bromo-4-chloro-3-indolyl-P-D-galactopyranoside (X-gal).
Results
Restriction enzyme analyses revealed the proper insertion of E. coli p-galactosidase-coding sequences into the multiple cloning site of pcDNA3.1/myc-His C. The functionality of the resulting constructs designated pcDNA3.1-cyt.lacZ and pcDNA3.1-nls.lacZ(+) was confirmed by X-gal staining of HeLa cells transfected with these recombinant plasmids. While pcDNA3.1-cyt.lacZ directed the synthesis of cytoplasmically located p-galactosidase molecules, the p-galactosidase protein encoded by pcDNA3.1-nls.lacZ(+) was predominantly detected in the cell nucleus.
Conclusion
The expression of cytoplasmic and nuclear variant of LacZ gene confirmed the ability of pcDNA3.1 as versatility nonviral vector for the experimental gene delivery study in mammalian cells

Keywords


1.Baum BJ, Goldsmith CM, Kok MR, Lodde BM, van Mello NM, Voutetakis A, et al. Advances in vector- mediated gene transfer. Immunol Lett 2003; 90:145-49.

2.Lechardeur D, Verkman AS, Lukacs GL (). Intracellular routing of plasmid DNA during non-viral gene transfer. Adv Drug Deliv Rev 2005; 57:755-767.

3.Peng S, Best SR, Hung CF, Loyo M, Lyford-Pike S, et al. Characterization of human papillomavirus type 11- specific immune responses in a preclinical model. Laryngoscope 2010; 120:504-510.

4.Liu F, Shollenberger LM, Huang L. Non-immunostimulatory nonviral vectors. FASEB J 2004; 18:1779-1781.

5.Johansson E, Wallgren P, Fuxler L, Domeika K, Lefevre F, Fossum C. The DNA vaccine vector pcDNA3 induces IFN-a production in pigs. Vet Immunol Immunopathol 2002; 87:29-40.

6.de Andrade GM, Machado RZ, Vidotto MC, Vidotto O. Immunization of bovines using a DNA vaccine (pcDNA3.1/MSP1b) prepared from the Jaboticabal strain of Anaplasma marginale. Ann N Y Acad Sci 2004; 1026:257-266.

7.Lu Y, Bai JG, Wang HH. Gene therapeutic treatment of pancreatic cancer based on injection of pcDNA3.1/CCK plasmid with xenogeneic homologous cholecystokinin. Exp Oncol 2006; 28:136-140.

8.Ye Z, Peng H, Fang Y, Feng J, Yang DS. The construction of the eukaryotic expression plasmid pcDNA3.1/azurin and the increased apoptosis of U2OS cells transfected with it. Cell Mol Biol Lett 2007; 12:407-421.

9.Ma HB, Wang XJ, Di ZL, Xia H, Li Z, Liu J, et al. Construction of targeted plasmid vector pcDNA3.1-Egr.1p- p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro. World J Gastroenterol 2007; 13:4214-4218. 

10.Goto H, Shuler FD, Lamsam C, Moller HD, Niyibizi C, et al. Transfer of lacZ marker gene to the meniscus. J Bone Joint Surg Am 1999; 81:918-925.

11.Kalnins A, Otto K, Ruther U, Muller-Hill B. Sequence of the lacZ gene of Escherichia coli. EMBO J 1983; 2:593-597.

12.Serebriiskii IG, Golemis EA. Uses of lacZ to study gene function: evaluation of beta-galactosidase assays employed in the yeast two-hybrid system. Anal Biochem 2000; 285:1-15.

13.Takahashi J, Itoh Y, Fujimori K, Imamura M, Wakayama Y, Miyagoe-Suzuki Y, et al. The utrophin promoter A drives high expression of the transgenic LacZ gene in liver, testis, colon, submandibular gland, and small intestine. J Gene Med 2005; 7:237-248.

14.Wang B, Lazaris A, Lindenbaum M, Stewart S, Co D, Perez C, et al. Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes. Mol Reprod Dev 2001; 60:433-438.

15.Nickerson HD, Colledge WH. A comparison of gene repair strategies in cell culture using a lacZ reporter system. Gene Ther 2003; 10:1584-1591.

16.Li L, Zemp RJ, Lungu G, Stoica G, Wang LV. Photoacoustic imaging of lacZ gene expression in vivo. J Biomed Opt 2007; 12:1-3.

17.Grant SG, Jessee J, Bloom FR, Hanahan D. Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A 1990; 87:4645-4649.

18.Kalderon D, Roberts BL, Richardson WD, Smith AE. A short amino acid sequence able to specify nuclear location. Cell 1984; 39:499-409.

19.Kalderon D, Richardson WD, Markham AF, Smith AE. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature 1984; 311:33-38.

20.Medina-Kauwe LK, Xie J, Hamm-Alvarez S. Intracellular trafficking of nonviral vectors. Gene Ther 2005; 12:1734-1751.

21.Darquet AM, Rangara R, Kreiss P, Schwartz B, Naimi S, Delaere P, et al. Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer. Gene Ther 1999; 6: 209-218.

22.Ziello JE, Huang Y, Jovin IS. Cellular endocytosis and gene delivery. Mol Med 2010; 16:222-29.

23.Asada M, Honda E, Imamura T. Construction of pcDNA3.1-based vectors with blasticidin and puromycin resistance markers. Anal Biochem 2006; 352:305-307.

24.Herrera E, Barcenas P, Hernandez R, Mendez A, Perez-Ishiwara G, Barron B. A 176 amino acid polypeptide derived from the mumps virus HN ectodomain shows immunological and biological properties similar to the HN protein. Virol J 2010; 7:195.

25.Ning JF, Zhu W, Xu JP, Zheng CY, Meng XL. Oral delivery of DNA vaccine encoding VP28 against white spot syndrome virus in crayfish by attenuated Salmonella typhimurium. Vaccine 2009; 27:1127-1135.

26.Capecchi MR. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 1980; 22:479-488.

27.Harraghy N, Gaussin A, Mermod N. Sustained transgene expression using MAR elements. Curr Gene Ther 2008; 8:353-366.

28.Gould DJ, Favorov P. Vectors for the treatment of autoimmune disease. Gene Ther 2003; 10:912-927.