Effective Targeted Gene Knockdown in Mammalian Cells Using the piggyBac Transposase-based Delivery System.

Nonviral gene delivery systems are rapidly becoming a desirable and applicable method to overexpress genes in various types of cells. We have recently developed a piggyBac transposase-based, helper-independent and self-inactivating delivery system (pmGENIE-3) capable of high-efficiency transfection of mammalian cells including human cells. In the following study, we have assessed the potential of this delivery system to drive the expression of short hairpin RNAs to knock down genes in human cells. Two independent pmGENIE-3 vectors were developed to specifically target knockdown of an endogenous gene, telomerase reverse transcriptase (TERT), in telomerase-positive human immortalized cell lines. As compared with a transposase-deficient vector, pmGENIE-3 showed significantly improved short-term transfection efficiency (~4-fold enhancement, 48 hours posttransfection) and long-term integration efficiency (~5-fold enhancement) following antibiotic selection. We detected a significant reduction of both TERT expression and telomerase activity in both HEK293 and MCF-7 breast carcinoma cells transfected with two pmGENIE-3 construct targeting distinct regions of TERT. Importantly, this knockdown of expression was sufficient to abrogate telomerase function since telomeres were significantly shortened (3-4 Kb, P < 0.001) in both TERT-targeted cell lines following antibiotic selection of stable integrants. Together, these data show the capacity of the piggyBac nonviral delivery system to stably knockdown gene expression in mammalian cells and indicate the potential to develop novel tumor-targeting therapies.Molecular Therapy-Nucleic Acids (2013) 2, e137; doi:10.1038/mtna.2013.61; published online 3 December 2013.

Short telomeres flirt with stem cell commitment.

High expression of telomerase in embryonic stem cells (ESCs) is important for their maintenance, but whether telomere length affects lineage commitment is unknown. In this issue of Cell stem cell, Pucci et al. (2013) reveal that ESCs with short telomeres exhibit unstable differentiation by inducing altered DNA methylation.

Cholinergic activation of hematopoietic stem cells: role in tobacco-related disease?

Tobacco use is associated with an increase in the white blood cell (WBC) count. This association has been attributed to bronchopulmonary inflammation and/or infection. It is not known if nicotine itself may play a role. The objective of this study was to determine whether nicotine itself could affect the WBC count, and to determine whether this was due to a direct effect on hematopoietic stem cells (HSC). C57Bl6J mice received nicotine orally, and measurements of the WBC count, bone marrow and spleen cellularity, and HSC count were made. To determine the functionality of HSCs, irradiated animals received bone marrow transplants from vehicle or nicotine-treated mice. Nicotine increased leukocytes in the peripheral blood, bone marrow and spleen. The peripheral red cell and platelet count were unaffected. Nicotine increased the frequency of HSC in the bone marrow. Isolated long-term HSCs from nicotine-treated mice transplanted into irradiated mice regenerated all hematopoietic cell lineages, demonstrating the functional competence of those HSCs. HSCs expressed nicotinic acetylcholine receptors (nAChRs), as documented by FITC-conjugated alpha-bungarotoxin binding. Nicotine increased soluble Kit ligand, consistent with stem cell activation. In conclusion, the data suggest a new mechanism for the increased WBC associated with tobacco use. The effect of nicotine to activate hematopoiesis may contribute to tobacco-related diseases.

Telomerase reverse transcriptase-dependent telomere equilibration mitigates tissue dysfunction in mTert heterozygotes.

Autosomal dominant mutations in telomere-associated factors elicit a disease known as dyskeratosis congenita (DKC), and patients suffer proliferative abnormalities associated with telomere erosion. Mice that are heterozygous for telomerase genes (Tert or Terc, hereafter referred to as mTert and mTerc) are useful models of telomerase haploinsufficiency, but do not strictly mimic DKC. In strains with long telomeres (>60 kbp), animals that are heterozygous for mTert undergo telomere erosion for nine generations and remain phenotypically normal. In an mTerc heterozygous strain with short telomeres (<15 kbp), early mortality arises after five to six generations, but dyskeratosis occurs only upon the further loss of mPot1b. We show that prolonged mTert heterozygosity (for greater than ten generations) did not elicit disease, even upon heterozygote interbreeding, and that telomeres reset to wild-type lengths. This lengthening did not occur in nullizygotes, and short telomeres inherited from mTert null parents were rescued only in heterozygous progeny. In the bone marrow, nullizygotes remained competent for radioprotection for three generations. Thus, gradual telomere erosion in the presence of telomerase may enable subsequent telomere extension, similar to that described in budding yeast. We speculate whether such adaptation occurs in normal human cells (or whether it could be induced in DKC-derived cells), and whether it might mitigate the impact of telomerase inhibition upon stem cells during cancer therapy.

Attenuated expression of SECIS binding protein 2 causes loss of telomeric reserve without affecting telomerase.

The family of selenoproteins have a broad range of functions, including protection against oxidative damage. Previous studies have shown that elevated levels of oxidative damage can induce accelerated loss of telomeric DNA during proliferation of mammalian cells. The incorporation of selenocysteine (Sec) into proteins in mammalian cells requires the Sec insertion sequence (SECIS) binding protein 2 (SBP2). Thus in the present study we have assessed the effect of knocking down the expression of SBP2 on telomere length. Following knock-down of SBP2 expression in two different human cell lines, the MSTO mesothelioma cell line ( approximately 5Kb average telomere length) and SY5Y neuroblastoma cell line (approximately 4.2Kb average telomere length), we observed a significant reduction (-0.6 to -1.1 Kb; P