Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells

Zeocin is a member of the bleomycin/phleomycin family of antibiotics, known to bind and cleave DNA. We established human SK-OV-3 cells that stably express the Zeocin resistance gene (Sh ble) using an ecdysone-inducible mammalian expression system. Surprisingly, our results demonstrated that Zeocin, added in the culture medium to maintain the expression of the ecdysone receptor, was responsible for the formation of DNA strand breaks in the recombinant cells. This suggests that the Zeocin is not completely detoxified and is still able to cleave DNA, despite the stable expression of the Sh ble gene in the recombinant clones. Our study indicates that one needs to be very cautious in the interpretation of data involving stable cell lines selected with Zeocin.     

GANP suppresses DNA recombination, measured by direct-repeat beta-galactosidase gene construct, but does not suppress the type of recombination applying to immunoglobulin genes in mammalian cells

Immunoglobulin V-region somatic hypermutation and C-region class-switch recombination are initiated by activation-induced cytidine deaminase (AID) in B-cells. AID-induced DNA damage at the immunoglobulin S-region is known to be repaired by non-homologous end-joining, but repair mechanisms at the V-region remain to be elucidated. In Saccharomyces cerevisiae, DNA homologous recombination is regulated by the expression of Sac3, involved in actin assembly, cell cycle transition and mRNA metabolism. Here, we demonstrate that the Sac3-homologue GANP suppresses DNA recombination in a direct-repeat beta-galactosidase gene construct in mammalian cells. Homozygous ganp gene knockout is embryonic lethal in mice. Embryonic fibroblasts immortalized from hetero-deficient ganp(+/-) mice showed more DNA recombination than wild-type. In contrast, over-expression of GANP suppressed either spontaneous DNA recombination or that caused by the introduction of aid cDNA into NIH3T3 cells (susceptible to I-sceI restriction enzyme cleavage but not to RAG-mediated immunoglobulin gene recombination). GANP suppresses the DNA recombination not only on the extrachromosomal DNA construct but also on the integrated DNA. The Sac3-homology portion is necessary for the suppressive activity, but the truncated carboxyl terminal MCM3-binding/acetylating region adversely augmented DNA recombination, acting as a dominant negative form. Expression of full-length GANP is critical for suppression of DNA hyper-recombination in mammalian cells.     

Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production

Forkhead box P3 (FOXP3) is currently thought to be the most specific marker for naturally occurring CD4(+)CD25(+) T regulatory cells (nTregs). In mice, expression of FoxP3 is strictly correlated with regulatory activity, whereas increasing evidence suggests that in humans, activated T effector cells (Teffs) may also express FOXP3. In order to better define the role of FOXP3 in human Teff cells, we investigated the intensity and kinetics of expression in ex vivo Teff cells, suppressed Teff cells and Teff cell lines. We found that all dividing Teff cells expressed FOXP3, but only transiently, and at levels that were significantly lower than those in suppressive nTregs. This temporary expression in Teff cells was insufficient to suppress expression of reported targets of FOXP3 repressor activity, including CD127, IL-2 and IFN-gamma, and was not correlated with induction of a nTreg phenotype. Thus expression of FOXP3 is a normal consequence of CD4(+) T cell activation and, in humans, it can no longer be used as an exclusive marker of nTregs. These data indicate that our current understanding of how FOXP3 contributes to immune tolerance in humans needs to be re-evaluated.     

Protein content does not correspond to DNA content in human cardiomyocytes

Department of Pathological Anatomy, Institute of Surgery, Academy of Medical Sciences of the USSR. Laboratory of Cytology, Institute of Developmental Biology, Academy of Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 111, No. 6, pp. 652–653, June, 1991.     

Thymic production of human FOXP3(+) regulatory T cells is stable but does not correlate with peripheral FOXP3 expression

In humans functionally mature FOXP3⁺ regulatory T (Treg) cells can be found already in the fetus, but the kinetics of their maturation is still unknown. Here, we show that from birth to until 10 years of age the thymic production of FOXP3⁺ Treg cells is very stable and correlates with T-lymphopoiesis in general. The level of FOXP3 expression in the blood was also very stable, even when children and adults were compared, but there was no correlation between thymic and peripheral FOXP3 levels. Analysis of the cell cycle-associated marker Ki67 showed that a substantial fraction of peripheral FOXP3⁺ cells is dividing. This characteristic was obtained in the periphery, since it was not observed in thymic CD4⁺ FOXP3⁺ cells. These data suggest that the thymic output of human Treg cells is intrinsically stable, while in the periphery the increased rate of proliferation severs the connection between production and homeostatic maintenance of the FOXP3⁺ Treg cell population.     

Efficient and stable transgene expression in human embryonic stem cells using transposon-mediated gene transfer

Efficient and stable genetic modification of human embryonic stem (ES) cells is required to realize the full scientific and potential therapeutic use of these cells. Currently, only limited success toward this goal has been achieved without using a viral vector. The Sleeping Beauty (SB) transposon system mediates nonviral gene insertion and stable expression in target cells and tissues. Here, we demonstrate use of the nonviral SB transposon system to effectively mediate stable gene transfer in human ES cells. Transposons encoding (a) green fluorescent protein coupled to the zeocin gene or (b) the firefly luciferase (luc) gene were effectively delivered to undifferentiated human ES cells with either a DNA or RNA source of transposase. Only human ES cells cotransfected with transposon- and transposase-encoding sequences exhibited transgene expression after 1 week in culture. Molecular analysis of transposon integrants indicated that 98% of stable gene transfer resulted from transposition. Stable luc expression was observed up to 5 months in human ES cells cotransfected with a transposon along with either DNA or RNA encoding SB transposase. Genetically engineered human ES cells demonstrated the ability to differentiate into teratomas in vivo and mature hematopoietic cells in vitro while maintaining stable transgene expression. We conclude that the SB transposon system provides an effective approach with several advantages for genetic manipulation and durable gene expression in human ES cells.     

同源无启动子DNA片段抑制人胰腺癌细胞株整合基因的表达

目的观察仅转染无启动子绿色荧光蛋白（GFP）的cDNA全长片段能否使整合有GFP基因的胰腺癌细胞内GFP表达降低。方法用pEGFP—C1质粒转染人胰腺癌细胞株PANC-1,G418筛选及流式细胞术分选建立GFP蛋白高表达的稳定细胞株。用PCR方法从pEGFP—C1扩增出GFP基因全长cDNA并将其克隆至无启动子的复制型质粒PUC19,得到重组质粒即PUC—GFP。实验分为4组：空白对照组、空质粒组（PUC组）、GFP重组质粒干扰组（PUC—GFP组）、GFP小RNA干扰组（siGFP组）。分别用稳定表达GFP和未经处理PANC-1细胞进行实验。用Western印迹、流式细胞术与倒置荧光显微镜检测重组质粒对细胞内稳定表达的GFP的影响及对GFP阴性细胞pEGFPC1质粒瞬时转染后绿色荧光表达强度的影响。结果PUC—GFP可使稳定细胞株内的GFP表达下降,并且呈剂量依赖性。PUC—GFP组绿色荧光强度减弱程度和空白对照及空质粒组差异有统计学意义（P〈0．05）和siGFP组差异无统计学意义（P〉0．05）。转染后第4天GFP表达降低并可持续至第6天,第4天后PUC—GFP组和siGFP组对GFP的抑制差异无统计学意义（P〉0．05）。PUC—GFP与pEGFP—C1共转染GFP阴性PANC-1细胞株可使pEGPC1的瞬时转染效率降低。这两种情况下,荧光降低程度均和转染PUC—GFP的量呈正相关。结论仅转染无启动子的某个特定基因的全长cDNA能使哺乳细胞内相应同源性基因的表达降低。DNA干扰可用于哺乳动物细胞。     

Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells

High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells. Disclosure of potential conflicts of interest is found at the end of this article.     

Tat protein expression in MDBK cells does not confer susceptibility to bovine immunodeficiency virus

Summary.  The ability of BIV strain R29 to infect bovine cell lines in the presence or absence of a functional lentiviral Tat protein is described. Jembrana disease virus (JDV) Tat protein was stably expressed in MDBK cells. No viral replication could be detected in this cell line after infection with BIV R29. Transfection of MDBK cells and MDBK Tat expressing cells with BIV R29 proviral DNA established that BIV R29 could not replicate in MDBK cells. Whether viral entry into MDBK cells is also a block to BIV R29 infection of MDBK cells has yet to be established. Received August 8, 2001 Accepted October 18, 2001     

Social stress does not alter the expression of sensitization to cocaine

The effects of chronic social stress on behavioral sensitization to cocaine were investigated in the Syrian hamster. Adolescent animals received either 15 mg/kg i.p. of cocaine or saline twice per day for 7 consecutive days. Two weeks following the last injection (young adulthood), they were given a challenge dose of 5 mg/kg i.p. of cocaine and scored for locomotion. Motor activity was significantly greater in cocaine-treated animals, demonstrating sensitization to this psychostimulant. Following the results of the first study, another group of adolescent animals was exposed to either a novel clean cage (control) or an aggressive resident male hamster (social stress) for 15 min following an injection of cocaine (20 mg/kg i.p. once daily) or saline for 7 consecutive days. The groups were as follows: Social Stress/Cocaine (SSC), No Social Stress/Cocaine (NSSC), Social Stress/Saline (SSS) and No Social Stress/Saline (NSSS). Two weeks following the last injection (Day 21), all animals were given a challenge dose of cocaine (5 mg/kg i.p.) and were rescored for locomotion. At that time, the suppressive effect of stress on locomotion was no longer detectable, as the expression of sensitization was observed in the NSSC but not in the SSC group. These results suggest that chronic social stress administered during adolescence does not cross-sensitize with cocaine in young adult hamsters.     

Biopsy of cleavage stage human embryos and diagnosis of single gene defects by DNA amplification.

Preimplantation diagnosis offers an alternative to current prenatal diagnostic techniques for patients known to be at risk of transmitting an inherited disease. One or two cells can be removed from cleavage stage embryos from patients undergoing in vitro fertilization. Affected embryos can be identified by analyzing the cells for the defect using the polymerase chain reaction. Following analysis, embryos identified as unaffected are replaced in the patient. The identification and transfer of female embryos was attempted in eight couples known to be at risk of transmitting various X-linked diseases. Five of the eight women became pregnant, two with twin and three with singleton pregnancies, after a total of 13 treatment cycles. Six of the seven fetuses were confirmed as being female after chorionic villus sampling. The prospects for the specific diagnosis of single gene defects are described.