Investigating the utility of the P1 Artificial Chromosome shuttle vector pJCPAC-Mam2 for gene therapy studies

Main Article Content

Jonathon Silow Coren


The goal of gene therapy is to deliver as many copies of a functional gene to a patient that lacks this protein. There have been some recent successes in rescuing some types of blindness and red blood cell disorders.  Current lentivirus-based vectors can only accommodate around 10 kilobases of foreign DNA.  Also, integration of the vector is random, so transcription of a given gene can vary several-fold depending on the chromosomal architecture where insertion occurs.  We constructed a P1 Artificial Chromosome (PAC) shuttle vector that has a greater than 150 kilobase insert size limit and remains as an episome in human cells.  We previously demonstrated that a PAC clone containing the p53 gene was transcribed and translated when transiently introduced into p53 homozygous null Saos-2 human osteosarcoma cells.  Furthermore, the apoptotic pathway was triggered in some of the cells.  A Saos-2 cell line that overexpresses the bcl-2 cDNA was generated using G418 selection so that the effects of different lipofection reagents on copy number and transcription and translation levels of p53 could be studied.  EndoFectin Max resulted in a several-fold higher transfection efficiency than Lipofectamine 3000.  Seven stable cell lines were generated from each transfection procedure using puromycin and G418 selection.  Unfortunately, only 7 of 13 cell lines contained measurable levels of p53 cDNA when total RNA was subjected to reverse transcriptase polymerase chain reaction (RT-PCR).  A PAC clone containing the green fluorescent gene from Pontellina plumata that is expressed from the strong cytomegalovirus promoter is being constructed to investigate whether Lipofectamine 3000 or EndoFectin Max maximizes transfection efficiency, plasmid copy number, and transcription and translation levels in the continuous cell lines HEK293, KG1 and Saos-2.

Article Details

How to Cite
COREN, Jonathon Silow. Investigating the utility of the P1 Artificial Chromosome shuttle vector pJCPAC-Mam2 for gene therapy studies. International Biology Review, [S.l.], v. 1, n. 1, may 2017. ISSN 2572-7168. Available at: <>. Date accessed: 13 nov. 2019. doi:
apoptosis, gene therapy, lipofection, transcription, translation


1. Maguire AM, Simonelli F, Pierce EA, Pugh EN Jr, Mingozzi F, Bennicelli J, Banfi S, Marshall KA, Testa F, Surace EM, Rossi S, Lyubarsky A, Arruda VR, Konkle B, Stone E, Sun J, Jacobs J, Dell'Osso L, Hertle R, Ma JX, Redmond TM, Zhu X, Hauck B, Zelenaia O, Shindler KS, Maguire MG, Wright JF, Volpe NJ, McDonnell JW, Auricchio A, High KA, Bennett J. Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med. 2008 May 22;358(21):2240-8. doi: 10.1056/NEJMoa0802315.

2. Tolmachova T, Wavre-Shapton ST, Barnard AR, MacLaren RE, Futter CE, Seabra MC. Retinal pigment epithelium defects accelerate photoreceptor degeneration in cell type-specific knockout mouse models of choroideremia. Invest Ophthalmol Vis Sci. 2010 Oct;51(10):4913-20. doi:10.1167/iovs.09-4892.

3. Ashtari M, Cyckowski LL, Monroe JF, Marshall KA, Chung DC, Auricchio A, Simonelli F, Leroy BP, Maguire AM, Shindler KS, Bennett J. The human visual cortex responds to gene therapy-mediated recovery of retinal function. J Clin Invest. 2011 Jun;121(6):2160-8. doi: 10.1172/JCI57377.

4. Weleber RG, Pennesi ME, Wilson DJ, Kaushal S, Erker LR, Jensen L, McBride MT, Flotte TR, Humphries M, Calcedo R, Hauswirth WW, Chulay JD, Stout JT. Results at 2 Years after Gene Therapy for RPE65-Deficient Leber Congenital Amaurosis and Severe Early-Childhood-Onset Retinal Dystrophy. Ophthalmology. 2016 Jul;123(7):1606-20. doi: 10.1016/j.ophtha.2016.03.003.

5. Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, Linch DC, Chowdary P, Riddell A, Pie AJ, Harrington C, O'Beirne J, Smith K, Pasi J, Glader B, Rustagi P, Ng CY, Kay MA, Zhou J, Spence Y, Morton CL, Allay J, Coleman J, Sleep S, Cunningham JM, Srivastava D, Basner-Tschakarjan E, Mingozzi F, High KA, Gray JT, Reiss UM, Nienhuis AW, Davidoff AM. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med. 2011 Dec 22;365(25):2357-65. doi:10.1056/NEJMoa1108046.

6. Nienhuis AW, Nathwani AC, Davidoff AM. Gene Therapy for Hemophilia. Hum Gene Ther. 2016 Apr;27(4):305-8. doi: 10.1089/hum.2016.018.

7. Breda L, Casu C, Gardenghi S, Bianchi N, Cartegni L, Narla M, Yazdanbakhsh K, Musso M, Manwani D, Little J, Gardner LB, Kleinert DA, Prus E, Fibach E, Grady RW, Giardina PJ, Gambari R, Rivella S. Therapeutic hemoglobin levels after gene transfer in β-thalassemia mice and in hematopoietic cells of β-thalassemia and sickle cells disease patients. PLoS One. 2012;7(3):e32345. doi: 10.1371/journal.pone.0032345.

8. Mansilla-Soto J, Riviere I, Boulad F, Sadelain M. Cell and Gene Therapy for the Beta-Thalassemias: Advances and Prospects. Hum Gene Ther. 2016 Apr;27(4):295-304. doi: 10.1089/hum.2016.037.

9. Ribeil JA, Hacein-Bey-Abina S, Payen E, Magnani A, Semeraro M, Magrin E, Caccavelli L, Neven B, Bourget P, El Nemer W, Bartolucci P, Weber L, Puy H, Meritet JF, Grevent D, Beuzard Y, Chrétien S, Lefebvre T, Ross RW, Negre O, Veres G, Sandler L, Soni S, de Montalembert M, Blanche S, Leboulch P, Cavazzana M. Gene Therapy in a Patient with Sickle Cell Disease. N Engl J Med. 2017 Mar 2;376(9):848-855. doi: 10.1056/NEJMoa1609677.

10. Gaspar HB, Cooray S, Gilmour KC, Parsley KL, Zhang F, Adams S, Bjorkegren E, Bayford J, Brown L, Davies EG, Veys P, Fairbanks L, Bordon V, Petropoulou T, Kinnon C, Thrasher AJ. Hematopoietic stem cell gene therapy for adenosine deaminase-deficient severe combined immunodeficiency leads to long-term immunological recovery and metabolic correction. Sci Transl Med. 2011 Aug 24;3(97):97ra80. doi: 10.1126/scitranslmed.3002716.

11. Cicalese MP, Ferrua F, Castagnaro L, Pajno R, Barzaghi F, Giannelli S3, Dionisio F, Brigida I, Bonopane M, Casiraghi M, Tabucchi A, Carlucci F, Grunebaum E, Adeli M, Bredius RG, Puck JM, Stepensky P, Tezcan I, Rolfe K, De Boever E, Reinhardt RR, Appleby J, Ciceri F, Roncarolo MG, Aiuti A. Update on the safety and efficacy of retroviral gene therapy for immunodeficiency due to adenosine deaminase deficiency. Blood. 2016 Jul 7;128(1):45-54. doi: 10.1182/blood-2016-01-688226.

12. Lukashev AN, Zamaytin AA Jr. Viral vectors for gene therapy: current state and clinical perspective. Biochemistry (Mosc). 2016 Jul;81(7):700-8. doi:

13. Gil-Farina I, Schmidt M. Interaction of vectors and parental viruses with the host genome. Curr Opin Virol. 2016 Dec;21:35-40. doi: 10.1016/j.coviro.2016.07.004.

14. Kattenhorn LM, Tipper CH, Stoica L, Geraghty DS, Wright TL, Clark KR, Wadsworth SC. Adeno-associated virus gene therapy for liver disease. Hum Gene Ther. 2016 Dec;27(12):947-961. doi: 10.1089/hum.2016.160.

15. Manson AL, Trezise AE, MacVinish LJ, Kasschau KD, Birchall N, Episkopou V, Vassaux G, Evans MJ, Colledge WH, Cuthbert AW, Huxley C. Complementation of null CF mice with a human CFTR YAC transgene. EMBO J. 1997 Jul 16;16(14):4238-49.

16. Sarsero JP, Li L, Holloway TP, Voullaire L, Gazeas S, Fowler KJ, Kirby DM, Thorburn DR, Galle A, Cheema S, Koenig M, Williamson R, Ioannou PA. Human BAC-mediated rescue of the Friedreich ataxia knockout mutation in transgenic mice. Mamm Genome. 2004 May;15(5):370-82.

17. Chatterjee PK, Shakes LA, Srivastava DK, Garland DM, Harewood KR, Moore KJ, Coren JS. Mutually exclusive recombination of wild-type and mutant loxP sites in vivo facilitates transposon-mediated deletions from both ends of genomic DNA in PACs. Nucleic Acids Res. 2004 Oct 19;32(18):5668-76.

18. Fuesler J, Nagahama Y, Szulewski J, Mundorff J, Bireley S, Coren JS. An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies and gene therapy. Gene. 2012 Apr 1;496(2):103-9. doi: 10.1016/j.gene.2012.01.011.

19. Marcellus RC, Teodoro JG, Charbonneau R, Shore GC, Branton PE. Expression of p53 in Saos-2 osteosarcoma cells induces apoptosis which can be inhibited by Bcl-2 or the adenovirus E1B-55 kDa protein. Cell Growth Differ. 1996 Dec;7(12):1643-50.

20. Diller L, Kassel J, Nelson CE, Gryka MA, Litwak G, Gebhardt M, Bressac B, Ozturk M, Baker SJ, Vogelstein B, et al. p53 functions as a cell cycle control protein in osteosarcomas. Mol Cell Biol. 1990 Nov;10(11):5772-81.

21. Yates JL, Warren N, Sugden B. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature. 1985 Feb 28-Mar 6;313(6005):812-5.

22. Donai K, Kuroda K, Guo Y, So KH, Sone H, Kobayashi M, Nishimori K, Fukuda T. Establishment of a reporter system to monitor silencing status in induced pluripotent stem cell lines. Anal Biochem. 2013 Dec 1;443(1):104-12. doi: 10.1016/j.ab.2013.08.014.

Most read articles by the same author(s)

Obs.: This plugin requires at least one statistics/report plugin to be enabled. If your statistics plugins provide more than one metric then please also select a main metric on the admin's site settings page and/or on the journal manager's settings pages.