Evaluation of BAC-Based Reporters for the Study of Human Telomerase
Open Access
- Author:
- Leiby, Melanie Ann
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 12, 2006
- Committee Members:
- Henry Joseph Donahue, Committee Chair/Co-Chair
Sarah Bronson, Committee Member
Kristin Ann Eckert, Committee Member
Mary Judith Tevethia, Committee Member - Keywords:
- bacterial artificial chromosomes
transcriptional regulation
telomerase
recombineering - Abstract:
- The ongoing efforts to sequence eukaryotic genomes have revealed that contrary to initial predictions, more complex organisms do not necessarily have more complex genomes or more genes. Instead, it has been shown that higher eukaryotes have more complex gene regulatory mechanisms, leading to increased organismal complexity. Understanding the ways in which genes are regulated is critical to understanding how organisms function in normal and diseased states. Many techniques and model systems have been used to study gene regulation, including transgenic mice and cell lines and promoter analysis via reporter genes. Some of the biggest problems when studying gene regulation are the susceptibility of transgenes to position effect variegation (PEV) and copy number dependence. To alleviate these problems, techniques have been developed to introduce transgenes in a single copy at predetermined genomic loci. Another concern when using standard transgenes to study gene regulation is that they do not always mimic the expression patterns of the endogenous locus, either because they do not contain all appropriate regulatory elements and/or are unable to re-form the native chromatin environment. The use of yeast, bacterial, and P1 artificial chromosomes (YACs, BACs, and PACs, respectively) as transgenes has been shown to increase the likelihood that a transgene will be expressed in an endogenous manner due to the increased amount of genomic sequence they harbor. Therefore, transgenics created using artificial chromosomes targeted in a single copy to a predetermined genomic locus should be very beneficial to the study of gene regulation. One gene that would benefit from such a system is human telomerase reverse transcriptase (TERT). TERT encodes a reverse transcriptase specific for telomeres, the tandemly repeated sequence located at the ends of all eukaryotic chromosomes that, in addition to their associated proteins, serve as caps to prevent critical genetic information from being lost due to the end replication problem and protect chromosomes from end-to-end fusions. In humans, TERT is expressed at high levels during embryogenesis, but in adult somatic cells, TERT is silenced; however, in 85-90% of all human tumors, telomerase is reactivated. The correlation between a lack of telomerase activity and hTERT mRNA seems to suggest telomerase expression is regulated predominantly at the level of transcription; however, despite almost 25 years of study, the mechanisms of hTERT regulation have remained elusive. One of the main reasons for this is the lack of a powerful model system. Mice, which are often used to study human genes, are not good models for studying hTERT because mTERT expression is much more promiscuous than that of hTERT and their overall telomere length is much longer. Additionally, transiently expressed, hTERT promoter-based reporters do not always mimic the endogenous telomerase activity of the cells into which they are introduced. We hypothesized that an hTERT reporter created from a BAC introduced in a single copy to a preexisting acceptor locus via recombinase-mediated cassette exchange (RMCE) would be able to mimic the endogenous telomerase expression patterns of genetically related telomerase positive (TERT(+)) and telomerase negative (TERT(-)) cells. To test this hypothesis, an acceptor locus containing a tkNeo fusion gene and Cre recombinase fused to the estrogen receptor under control of the cytomegalovirus immediate early promoter and the Cre recombinase recognition sites loxP and lox511 surrounded by single copies of the chicken ƒÒ-globin insulator, cHS4, was created and introduced into genetically related TERT(+) and (-) cells via retroviral integration. (These TERT(+) and (-) cells were immortal clones that arose after IMR-90 human fibroblasts stably expressing SV40 large T and small t antigens survived crisis.) Overall, five TERT(+) and three TERT(-) cell lines containing the acceptor locus at a single site were identified. Several BAC reporters were constructed by inserting the firefly, Renilla, and hRenilla luciferase open reading frames into the start codons of CRR9 and TERT, as well as the puromycin resistance gene (puro) into the vector backbone, of the hTERT-containing BAC RP11-117B23 using a two-step homologous recombination technique in bacteria termed recombineering. Smaller RMCE-competent plasmids were also created using recombineering by introducing puro into the base BAC vectors. The ability of the acceptor locus-containing TERT(+) and TERT(-) cells to undergo RMCE was verified using the plasmid-based BAC vectors. Attempts to target both circular and linear hTERT-containing BAC reporters to the acceptor locus were unsuccessful in both TERT(+) and (-) cells. Random integration of linearized BAC reporters to the genome of acceptor locus-containing TERT(+) and (-) cells resulted in a total of 186 stable integrants. Analysis of these clones by luciferase assay, Southern hybridization, and PCR amplification revealed that only 12 of these 186 clones (6.45%) appeared to be intact after integration and were therefore useful for addressing the modified hypothesis that BAC-based hTERT reporters would be able to mimic endogenous telomerase expression upon their introduction to genetically related TERT(+) and (-) cells. All 12 of these clones (seven TERT(+) and five TERT(-)) expressed high-level CRR9 expression, as read out by firefly luciferase activity, and low-level TERT expression, as read out by Renilla luciferase activity. Therefore, BAC-based hTERT reporters do not appear to mimic endogenous telomerase expression patterns in genetically related, crisis-derived TERT(+) and (-) human fibroblasts.