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1.
Nechushtan H Pham D Zhang Y Morgensztern D Yi KH Shin SU Federoff HJ Bowers WJ Tolba KA Rosenblatt JD 《Cancer immunology, immunotherapy : CII》2008,57(5):663-675
Treatment of cancer with cytotoxic agents may induce lymphopenia. Adoptively transferred T cells have been reported to display enhanced anti-tumor efficacy in the lymphopenic setting. We reasoned that the anti-tumor effects of adoptively transferred cells in the lymphopenic host could be further augmented through local provision of an innate stimulus in the tumor bed. Utilizing a model in which mice were irradiated to induce lymphopenia, with limited shielding to allow tumor growth, we demonstrate that “triple” therapy consisting of radiation-induced lymphopenia, adoptive transfer of naïve CD8+ T cells, and intra-tumoral HSV amplicon injection resulted in reduced tumor growth compared to the combination of any two of the aforementioned interventions. To gain insight into the mechanism underlying this effect we studied the effects of HSV amplicon transduction into tumors on cytokine expression and on anti-tumor specific T cells. HSV amplicon transduction specifically induced several cytokine mRNAs including IFN-γ, and IP-10. Adoptively transferred transgenic OT-1 T cells directed against Ovalbumin were more effective against Ovalbumin-expressing tumors when combined with intra-tumoral HSV amplicon injections in the lymphopenic host. Following intra-tumoral HSV-amplicon injections, anti-tumor T cells secreted higher levels of interferon-γ in response to in-vitro re-stimulation with tumor cells, implying that HSV amplicon injection provided a strong signal for T cell activation. Combining adoptive transfer of naïve T cells in the lymphopenic setting with local T cell stimulation may facilitate expansion and activation of anti-tumor T cell populations in vivo, resulting in enhanced anti-tumor responses without the need to resort to prolonged in vitro T cell culture and/or manipulation. 相似文献
2.
Treatment of medullary thyroid carcinoma by combined expression of suicide and interleukin-2 genes 总被引:7,自引:0,他引:7
M. N. Soler G. Milhaud F. Lekmine F. Treilhou-Lahille D. Klatzmann S. Lausson 《Cancer immunology, immunotherapy : CII》1999,48(2-3):91-99
Inherited medullary thyroid carcinomas (MTC) are aggressive and resistant to conventional chemo- and radiotherapies. We evaluated
a novel strategy for treatment of MTC, combining “suicide” and interleukin-2 (IL-2) gene therapies. Tumors were produced in
Wag/Rij rats by orthotopic injection of the rMTC 6–23 cell line, and/or derivatives expressing the herpes simplex virus 1
thymidine kinase (HSV1-TK) gene (rMTC-TK). Ganciclovir, a nucleoside analog selectively transformed to a toxic metabolite
by HSV1-TK, totally eradicated rMTC-TK tumors in 60% of the animals. 1:1 rMTC and rMTC-TK mixed tumors were also strongly
inhibited by ganciclovir (P < 0.05), indicating the occurrence of an efficient “bystander” effect in vivo. Double labelling of rMTC cell membranes and
apoptotic nuclei revealed that, as with the TK+ cells, some TK− cells died by apoptosis. A 1:1 mixture of rMTC and rMTC-TK cells was administered to produce established tumors and then
rMTC cells, transfected to express the IL-2 gene (rMTC-IL2), were inoculated. Combined ganciclovir and IL-2 treatment improved
the inhibition of tumor growth compared to that following ganciclovir alone (86% compared to 54%, P < 0.05). This treatment also significantly enhanced macrophage activation and tumor infiltration by CD8+ and CD4+ T lymphocytes. These results open an avenue for combining suicide and immunoregulatory gene therapies for MTC management
in man.
Received: 1 October 1998 / Accepted: 1 January 1999 相似文献
3.
4.
Kim SJ Sadelain M Lee JS Seong RH Yun YS Jang YJ Chung HY 《Cancer immunology, immunotherapy : CII》1999,47(5):257-264
We show that the tumor-specific primary cytotoxic T lymphocytes (CTL) induced in vitro with the MCA205 fibrosarcoma cells
transduced with the B7.1 (CD80) gene are highly effective in adoptive-transfer therapy of the parental tumors. The MCA205
fibrosarcoma cell line was transduced with the retroviral vectors encoding the B7.1 gene and tested for their efficiency as
stimulators in short-term (5 days) mixed lymphocyte/tumor cell cultures with highly purified syngenic, unprimed T cells as
responders. The induction of the CTL required the presence of a low dose of interleukin-2 (25 U/ml). The injection of the
CTL prevented colony formation by the intravenously injected tumor cells in a lung colonization assay in which the CTL were
injected after inoculation of tumor cells. We also showed that the adoptive transfer of the same T cells was effective in
delaying the growth of the subcutaneously injected tumor cells. These results imply that the short-term mixed lymphocyte/tumor
cell culture with the tumor cells transduced with the gene for the B7.1 costimulatory molecule is potentially a good source
of CTL for adoptive-transfer therapy of tumors.
Received: 30 June 1998 / Accepted: 5 August 1998 相似文献
5.
Herpes simplex virus (HSV)-mediated ICAM-1 gene transfer abrogates tumorigenicity and induces anti-tumor immunity.
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M. D''''Angelica C. Tung P. Allen M. Halterman K. Delman T. Delohery D. Klimstra M. Brownlee H. Federoff Y. Fong 《Molecular medicine (Cambridge, Mass.)》1999,5(9):606-616
BACKGROUND: Costimulatory and cellular adhesion molecules are thought to be essential components of antigen presentation in the immune response to cancer. The current studies examine gene transfer utilizing herpes viral amplicon vectors (HSV) to direct surface expression of adhesion molecules, and specifically evaluate the potential of a tumor-expressing intercellular adhesion molecule-1 (ICAM-1) to elicit an anti-tumor response. MATERIALS AND METHODS: The human ICAM-1 (hICAM1) gene was inserted into an HSV amplicon vector and tested in a transplantable rat hepatocellular carcinoma and in a human colorectal cancer cell line. Cell surface ICAM-1 expression was assessed by flow cytometry. Lymphocyte binding to HSV-hICAM1-transduced cells was compared with that to cells transduced with HSV not carrying the ICAM gene. Tumorigenicity of HSV-hICAM1-transduced tumor cells were tested in syngeneic Buffalo rats. Additionally, immunization with irradiated (10,000 rads) HSV-hICAM1-transduced tumor cells was performed to determine its effect on tumor growth. RESULTS: A 20-min exposure of tumor cells at a multiplicity of infection (MOI) of 1 resulted in high-level cell surface expression of human ICAM in approximately 25% of tumor cells. Transduced rat or human tumor cells exhibited significantly enhanced binding of lymphocytes (p < 0.05). HSV-hICAM1-transduced cells elicited an increase in infiltration by CD4(+) lymphocytes in vivo and exhibited decreased tumorigenicity. Immunization with irradiated HSV-hICAM1-transduced cells protected against growth of subsequent injected parental tumor cells. CONCLUSIONS: HSV amplicon-mediated gene transfer is an efficient method for modifying the cell surface expression of adhesion molecules. Increased tumor expression of ICAM-1 represents a promising immune anti-cancer strategy. 相似文献
6.
Zager JS Delman KA Malhotra S Ebright MI Bennett JJ Kates T Halterman M Federoff H Fong Y 《Molecular medicine (Cambridge, Mass.)》2001,7(8):561-568
BACKGROUND: Herpes simplex type I (HSV)-based vectors have been used experimentally for suicide gene therapy, immunomodulatory gene delivery, and direct oncolytic therapy. The current study utilizes the novel concept of regional delivery of an oncolytic virus in combination with or serving as the helper virus for packaging herpes-based amplicon vectors carrying a cytokine transgene, with the goal of identifying if this combination is more efficacious than either modality alone. MATERIALS AND METHODS: A replication competent oncolytic HSV (G207) and a replication incompetent HSV amplicon carrying the gene for the immunomodulatory cytokine IL-2 (HSV-IL2) were tested in murine syngeneic colorectal carcinoma and in rat hepatocellular carcinoma models. Liver tumors were treated with vascular delivery of (1) phosphate-buffered saline (PBS), (2) G207, (3) HSV-IL2, (4) G207 and HSV-IL2 mixed in combination (mG207/HSV- IL2), and (5) G207 as the helper virus for packaging the construct HSV-IL2 (pG207/HSV-IL2). RESULTS: Tumor burden was significantly reduced in all treatment groups in both rats and mice treated with high-dose G207, HSV-IL2, or both (p < 0.02). When a low dose of virus was used in mice, anti-tumor efficacy was improved by use of G207 and HSV-IL2 in combination or with HSV-IL2 packaged by G207 (p < 0.001). This improvement was abolished when CD4(+) and CD8(+) lymphocytes were depleted, implying that the enhanced anti-tumor response to low-dose combined therapy is immune mediated. CONCLUSIONS: Vascular regional delivery of oncolytic and amplicon HSV vectors can be used to induce improved anti-tumor efficacy by combining oncolytic and immunostimulatory strategies. 相似文献
7.
BACKGROUND: Firefly luciferase (Fluc) has routinely been used to quantitate and analyze gene expression in vitro by measuring the photons emitted after the addition of ATP and luciferin to a test sample. It is now possible to replace luminometer-based analysis of luciferase activity and measure luciferase activity delivered by viral vectors directly in live animals over time using digital imaging techniques. METHODS: An HSV amplicon vector expressing Fluc cDNA from an inducible promoter was delivered to cells in culture and into the mouse brain. In culture, expression of Fluc was measured after induction in a dose-dependent manner by a biochemical assay, and then confirmed by Western blot analysis and digital imaging. The vectors were then stereotactically injected into the mouse brain and Fluc expression measured non-invasively using bioluminescence imaging. RESULTS: Rapamycin-mediated induction of Fluc from an HSV amplicon vector in culture resulted in dose-dependent expression of Fluc when measured using a luminometer and by digital analysis. In mouse cortex, a single injection of an HSV amplicon vector (2 microl, 1x10(8) transducing units (t.u.)/ml) expressing Fluc from a viral promoter (CMV) was sufficient to detect robust luciferase activity for at least 1 week. Similarly, an HSV amplicon vector expressing Fluc under an inducible promoter was also detectable in the mouse cortex after a single dose (2 microl, 1x10(8) t.u./ml) for up to 5 days, with no detectable signal in the uninduced state. CONCLUSIONS: This HSV amplicon vector-based system allows for fast, non-invasive, semi-quantitative analysis of gene expression in the brain. 相似文献
8.
The transfer of naked deoxyribonucleic acid (DNA) represents an alternative to viral and liposomal gene transfer technologies
for gene therapy applications. Various procedures are employed to deliver naked DNA into the desired cells or tissues in vitro
and in vivo, such as by simple needle injection, particle bombardment, in vivo electroporation or jet injection. Among the
various nonviral gene delivery technologies jet injection is gaining increasing acceptance because it allows gene transfer
into different tissues with deeper penetration of the applied naked DNA. The versatile hand-held Swiss jet injector uses pressurized
air to force small volumes of 3 to 10 μL of naked DNA into targeted tissues. The β-galactosidase (LacZ) reporter gene construct and tumor necrosis factor α gene-expressing vectors were successfully jet injected at a pressure
of 3.0 bar into xenotransplanted human tumor models of colon carcinoma. Qualitative and quantitative expression analysis of
jet injected tumor tissues revealed the efficient expression of these genes in the tumors. Using this Swiss jet-injector prototype
repeated jet injections of low volumes (3–10 μL) into one target tissue can easily be performed. The key parameters of in
vivo jet injection such as jet injection volume, pressure, jet penetration into the tumor tissue, DNA stability have been
defined for optimized nonviral gene therapy. These studies demonstrate the applicability of the jet injection technology for
the efficient and simultaneous in vivo gene transfer of two different plasmid DNAs into tumors. It can be employed for nonviral
gene therapy of cancer using minimal amounts of naked DNA. 相似文献
9.
Quanzhi Li Wendy Hudson Duo Wang Erica Berven Fatih M. Uckun John H. Kersey 《Cancer immunology, immunotherapy : CII》1998,47(3):121-130
The comparative advantages and disadvantages of intact antibodies and single-chain Fv as immunotoxins and radioimmunoconjugates
have been widely discussed but not directly compared. In this study, the in vivo properties of anti-CD19 B43 monoclonal antibody
and its derived single-chain Fv (FVS191) were studied in athymic nude mice bearing CD19-positive human lymphomas. B43 mab
and FVS191 were labeled with iodine-125 using iodine-beads, and immunoreactivities were determined to be 57% and 72%, respectively.
Scatchard analysis showed a similar high affinity for both. The results of pharmacokinetic studies revealed that FVS191 had
a rapid biphasic clearance from the circulation (T1/2α = 2.5 min, T1/2β = 3.7 h); The T1/2α and T1/2β phases of B43 mab were
determined to be 0.72 h and 57 h respectively. Biodistribution studies compared the uptake of labeled antibodies by CD19-positive
and by CD19-negative tumors. The peak percentages of injected dose were 5.7% at 12 h for B43 and 2.45% at 1 h for FVS191.
Radiolocalization indices (RI) demonstrated tumor-specific uptake for both, but higher uptake for B43. The optimal RI was
seen at 15 min for FVS191 and 6 h for B43. FVS191 was unstable in vivo, approximately 50% of the injected dose being degraded
in blood in 100 min. Radioactivity detected in the urine was present mainly as the deiodinized form of FVS191. The results
suggest that B43 mab is favored over FVS191 in biodistribution properties and in vivo stability. Because B43 Mab showed early
tumor-specific uptake, high RI values, and favorable tissue-to-blood ratios, it is a potential candidate for radioimmunotherapy
and immunotoxin therapy of B-cell leukemia and lymphoma.
Received: 17 June 1997 / Accepted: 17 June 1998 相似文献
10.
There is an enormous initiative to establish the genetic basis for disorders of brain function. Unfortunately, genetic intervention is not accomplished easily in the nervous system. One strategy is to engineer and deliver to neurons specialized viral vectors that carry a gene (or genes) of interest, thereby exploiting the natural ability of viruses to insert genetic material into cells. When delivered to brain cells, these vectors cause infected cells to increase the expression of the genes of interest. The ability to deliver genes into neurons in vitro and in vivo with herpes simplex virus (HSV) amplicon vectors has made it possible to carry out exactly these sorts of experiments. This technology has the potential to offer new insights into the etiology of a wide variety of neuropsychiatric disorders. We describe the use of HSV amplicon vectors to study Alzheimer disease, drug addiction, and depression, and discuss the considerations that enter into the use of these vectors both in vitro and in vivo. The HSV amplicon virus is a user-friendly vector for the delivery of genes into neurons that has come of age for the study of brain function. 相似文献
11.
The effects of DL-AP5 and glutamate on ghrelin-induced feeding behavior in 3-h food-deprived broiler cockerels 总被引:2,自引:0,他引:2
This study was designed to examine the effects of intracerebroventricular injection of DL-AP5 (N-methyl-D-aspartate (NMDA) receptor antagonist) and glutamate on ghrelin-induced feeding behavior in 3-h food-deprived (FD3) broiler
cockerels. At first, guide cannula was surgically implanted in the right lateral ventricle of chickens. In experiment 1, birds
were intracerebroventricularly injected with 0, 2.5, 5, and 10 nmol of DL-AP5. In experiment 2, chickens received 5 nmol DL-AP5
prior to the injection of 0.6 nmol ghrelin. In experiment 3, birds were administered with 0.6 nmol ghrelin after 300 nmol
glutamate, and the cumulative feed intake was determined at 3-h postinjection. The results of this study showed that the intracerebroventricular
injection of DL-AP5 increased food consumption in FD3 broiler cockerels (P ≤ 0.05), and this increase occurs in a dose-dependent manner. Moreover, the decreased food intake induced with the intracerebroventricular
injection of ghrelin was additively enhanced by pretreatment with glutamate, and this effect was attenuated by DL-AP5 administration(P ≤ 0.05).These results suggest that there is an interaction between ghrelin and glutamatergic system (through NMDA receptor)
on food intake in broiler cockerels. 相似文献
12.
Suresh K. Gupta Vivekananthan Kalaiselvan Sushma Srivastava Rohit Saxena Shyam S. Agrawal 《Biological trace element research》2010,136(3):258-268
Cataract is the opacification in eye lens and leads to 50% of blindness worldwide. The present study was undertaken to evaluate
the anticataract potential of Trigonella foenum-graecum Linn seeds (fenugreek) in selenite-induced in vitro and in vivo cataract. In vitro enucleated rat lenses were maintained
in organ culture containing Dulbecco’s modified Eagles medium (DMEM) alone or in addition with 100 μM selenite and served
as the normal and control groups, respectively. For the test group, the medium was supplemented with selenite and T. foenum-graecum aqueous extract. The lenses were incubated for 24 h at 37°C. After incubation, the lenses were processed for the estimation
of reduced glutathione (GSH), lipid peroxidation product (malondialdehyde), and the antioxidant enzymes. In vivo selenite
cataract was induced in 9-day-old rats by subcutaneous injection of sodium selenite (25 μmol/kg body weight). Animals in the
test group were injected with different doses of aqueous extract of T. foenum-graecum 4 h before the selenite challenge. A fall in GSH and a rise in malondialdehyde levels were observed in control as compared
to normal lenses. T. foenum-graecum significantly (P < 0.01) restored glutathione and decreased malondialdehyde levels. A significant restoration in the activities of antioxidant
enzymes such as superoxide dismutase (P < 0.01), catalase, (P < 0.01), glutathione peroxidase (P < 0.01), and glutathione-S-transferase (P < 0.01) was observed in the T. foenum-graecum supplemented group as compared to control. In vivo, none of the eyes was found with nuclear cataract in treated group as
opposed to 72.5% in the control group. T. foenum-graecum protects against experimental cataract by virtue of its antioxidant properties. Further studies are warranted to explore
its role in human cataract. 相似文献
13.
M. L. Rúa H. Atomi C. Schmidt-Dannert R. D. Schmid 《Applied microbiology and biotechnology》1998,49(4):405-410
An efficient expression system for the previously only weakly expressed thermophilic lipase BTL2 (Bacillus thermocatenulatus lipase 2) was developed for the production of large amounts of lipase in Escherichia coli. Therefore, the gene was subcloned in the pCYTEXP1 (pT1) expression vector downstream of the temperature-inducible λ promoter
PL. Three different expression vectors were constructed: (i) pT1-BTL2 containing the mature lipase gene, (ii) pT1-preBTL2 containing the prelipase gene and (iii) pT1-OmpABTL2 containing the mature lipase gene fused to the signal peptide of the OmpA protein, the major outer membrane
protein of E. coli. With pT1-BTL2 and pT1-preBTL2, comparable expression levels of 7000–9000 U/g cells were obtained independently of the E. coli host. In contrast, with E. coli JM105 harbouring pT1-OmpABTL2, 660 000 soluble lipase U/g cells was produced, whereas, with E. coli DH5α and BL321, production levels of 30 000 U/g cells were achieved. However, most of the lipase remained insoluble but active
after cell breakage because of the unprocessed OmpA signal peptide. A simple cholate extraction followed by proteinase K cleavage
and ultrafiltration allowed the isolation of 1.15 × 106 units of 90% pure mature lipase/wet cells.
Received: 29 August 1997 / Received revision: 17 November 1997 / Accepted: 18 November 1997 相似文献
14.
Ø. Tøien J. B. Mercer 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1998,168(2):73-80
Hypothalamic temperature (T
hypo) and metabolic heat production (M) were measured in seven conscious rabbits injected intravenously with either saline or
with Staphylococcus aureus, (8 · 107 cell walls · kg−1) while being subjected to a 3-h period of ramp-like total body cooling using a chronically implanted intravascular heat exchanger.
In pyrogen-injected animals cooling started (1) at the time of injection or (2) 70 min after injection. In (1) the fall in
T
hypo induced by heat extraction was similar (1.0 °C) in afebrile and febrile animals. In (2) there was a transient increase in
T
hypo of about 0.5 °C at a time corresponding to the start of fever resulting in a significantly smaller fall in T
hypo at the end of the 3-h cooling period (0.5 °C vs 0.9 °C, P < 0.05, n = 5). At this time in both (1) and (2) M was lower than theoretically expected from the increase in shivering threshold during
fever. However, most of this effect can be explained when available data showing a decrease in thermosensitivity during S. aureus-induced fever are taken into account. After cessation of cooling in both groups of febrile animals T
hypo rose to about 1 °C higher than the precooling level, which is comparable to the fever level in a separate series of experiments
with S. aureus injection without cooling (1.2 °C).
Accepted: 23 September 1997 相似文献
15.
Sakurai T Misawa E Yamada M Hayasawa H Motoyoshi K 《Cancer immunology, immunotherapy : CII》2000,49(2):94-100
We injected cyclophosphamide into mice and examined their natural killer (NK) activity both in vitro and in vivo. Cyclophosphamide
injection temporarily abrogated the lung clearance activity of Yac-1 lymphoma cells, which is considered to be an index of
NK activity in vivo. However, administration of recombinant human macrophage-colony-stimulating-factor (rhM-CSF) to cyclophosphamide-injected
mice restored the lung clearance activity. To clarify whether the administration of rhM-CSF activated NK cells, we purified
NK1.1+ cells from mice treated with cyclophosphamide and/or rhM-CSF and examined their functions (cytotoxicity, proliferation, and
interferon γ production) in vitro. Cyclophosphamide injection decreased the number, but did not suppress the functions of
NK1.1+ cells. The numbers of NK1.1+ cells in cyclophosphamide-injected mice restored by rhM-CSF administration. And the functions of NK1.1+ cells from both saline-injected and cyclophosphamide-injected mice were accelerated by rhM-CSF administration. These results
suggested that the temporary abrogation of NK activity in vivo caused by cyclophosphamide injection was due to a decrease
in the number and not to suppression of the functions of NK1.1+ cells. The injection of cyclophosphamide into mice increased the number of tumor (B16 melanoma) nodules formed in the lungs
and liver. However, treatment with rhM-CSF recovered the anti-metastatic activity in the lungs of cyclophosphamide-injected
mice. These results show that administration of rhM-CSF restores NK activity suppressed by cyclophosphamide injection in vivo.
Received: 28 September 1999 / Accepted: 23 December 1999 相似文献
16.
A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the
minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (cat
GC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0 × 10−7 and 4.7 × 10−7 transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous
replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly,
both shuttle vectors could also be mobilized efficiently from E. coli into different H.␣pylori recipients, with pHel2 showing an efficiency of 2.0 × 10−5 transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation
of a recA-deficient H. pylori mutant by the cloned H. pylorirecA
+ gene, and the expression of the heterologous green fluorescent protein (GFP) in H.␣pylori demonstrate the general usefulness of␣this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future.
Received: 22 April 1997 / Accepted: 4 November 1997 相似文献
17.
Peng QP Cao SF Lyu QF Xue SG Jin W Liu XY Zhang WJ Nielsen HI Kuang YP 《In vitro cellular & developmental biology. Animal》2011,47(8):565-572
The purpose of this study is to develop a novel method for the cryopreservation and efficient post-thaw recovery of individual
or small numbers of human spermatozoa. Spermatozoa equilibrated in cryoprotectant buffer were injected with an intracytoplasmic
sperm injection (ICSI) needle into a droplet of cryoprotectant on a homemade cryoleaf. The droplet was of cryoprotectant and
seminal plasma at a ratio of 1:1. The sperm-loaded cryoleaf was slowly lowered over and stored in liquid nitrogen. Spermatozoa
were thawed in a 37°C oil bath without dilution and centrifugation. To test the fertilizing ability of these spermatozoa,
the recovered spermatozoa were injected by ICSI into 1-d-old or in vitro-matured human oocytes. Fresh spermatozoa from the
same semen samples served as controls. The trials were performed in two separate experiments. In the first set of experiments,
92 spermatozoa were thawed and carefully investigated. The spermatozoa from percutaneous epididymal sperm aspiration had a
motility recovery of 92.9% (13/14); ejaculated spermatozoa had a motility recovery of 61.5% (48/78), and only 1.3% (1/78)
was lost. Together in the first and second set of experiments, the fertilization rates for the fresh and frozen–thawed spermatozoa
were 67.6% (25/37) and 60.6% (40/66), respectively (P = 0.052). The mean embryo cleavage rates in the fresh and frozen–thawed groups were 88% (22/25) and 85% (34/40), respectively
(P = 0.990). This cryopreservation method for individual or small numbers of human spermatozoa was efficient and simple. These
findings make this method a promising technique for the clinical application of ejaculated sperm from oligozoospermic patients. 相似文献
18.
Efficient transformation of pBR322 and its derived
plasmids, which have been widely used as cloning vectors in Escherichia
coli, was observed in Pseudomonas avenae (K1), the pathogen of
leaf blight disease in cereals. Moreover, there was a 10- to 50-fold
transformation efficiency (1.3–3.0 × 106/μg DNA) in the
proline-auxotrophic mutant (Pr47), whose virulence to rice seedlings
decreased. Similar enhancement of the frequency of transfer by mobilization
of RSF1010, a broad host range plasmid, was observed in the recipient Pr47
strain in mating with donor Pseudomonas syringae. The plasmids
harbored in these strains were maintained very stably after subcultures.
Thus, a highly efficient transformation system with pBR322-derived plasmids
used as a vector and Pseudomonas as a host bacterium was developed.
Received: 13 July 1996 / Accepted: 26 August 1996 相似文献
19.
S. E. J. Fischer H. G. A. M. van Luenen R. H. A. Plasterk 《Molecular & general genetics : MGG》1999,262(2):268-274
The Caenorhabditis elegans transposons Tc1 and Tc3 are able to transpose in heterologous systems such as human cell lines and zebrafish. Because these
transposons might be useful vectors for transgenesis and mutagenesis of diverse species, we determined the minimal cis requirements for transposition. Deletion mapping of the transposon ends shows that fewer than 100 bp are sufficient for transposition
of Tc3. Unlike Tc1, Tc3 has a second, internal transposase binding site at each transposon end. We found that these binding
sites play no major role in the transposition reaction, since they can be deleted without reduction of the transposition frequency.
Site-directed mutagenesis was performed on the conserved terminal base pairs at the Tc3 ends. The four terminal base pairs
at the ends of the Tc3 inverted repeats were shown to be required for efficient transposition. Finally, increasing the length
of the transposon from 1.9 kb to 12.5 kb reduced the transposition frequency by 20-fold, both in vivo and in vitro.
Received: 21 April 1999 / Accepted: 10 June 1999 相似文献
20.
Positive control 1 (PC1) (n = 9) goats were injected transthoracically into the left lung with live Pasteurella haemolytica biovar A, serovar 1 (PhA1) in polyacrylate (PA) beads on days 0 and 21. Positive control 2 (PC2) (n = 6) goats were nebulized
with live PhA1 and PA beads on days 0 and 21. Negative control (NC) goats (n = 6) were each injected transthoracically into
the left lung with PA beads alone on days 0 and 21. Four groups (n = 6) were administered PA beads mixed with ultraviolet
(UV) killed PhA1 on days 0 and 21. The treatment doses of bacteria for these groups were principal group 1 (PR1) injected
into the left lung (7.7 × 1010 cfu); PR2, 7.7 × 1010 UV-killed PhA1 injected subcutaneously (SC); PR3, 7.7 × 1010 UV-killed PhA1 injected SC only on day 21; PR4, nebulized with PA beads mixed with 5.6 × 1010 cfu of UV-killed PhA1; and PR5, nebulized with PA beads mixed with 5 × 108 cfu of UV-killed PhA1. All goats were challenged transthoracically in the right lung with 1 × 108 cfu of live PhA1 on day 42 and necropsied on day 46. The sizes of consolidated lung lesions at the challenge site were used
as a measure of immunity. The data show that the introduction of live PhA1 into the lungs of goats, either by injection or
aerosolization, offers excellent protection against a subsequent homologous challenge. The data also demonstrate that two
transthoracic injections (21 days apart) of UV-killed PhA1 (PR1), and subcutaneous injection of UV-killed PhA1(PR2) also offer
excellent protection against a subsequent homologous live PhA1 challenge. One SC injection of UV-killed PhA1 (PR3) appears
to offer only partial protection against a subsequent homologous live PhA1 challenge. Inhalation of UV-killed PhA1 mixed with
PA beads (PR4 and PR5) induced no protection in goats against a subsequent live PhA1 transthoracic challenge.
Received: 3 February 1998 / Accepted: 18 March 1998 相似文献