Characterizing proteases in an Antarctic Janthinobacterium sp. isolate: Evidence of a protease horizontal gene transfer event

We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium （named AU 11）, from a water sample collected in Lake Uruguay （King George Island, South Shetlands）. AUI 1 （growth between 4℃ and 30℃） produces a single cold-active extracellular protease （ExPAU11）, differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-ToF MS） as an alkaline metallo-protease （70% coverage with an extracellular protease of Janthinobacterium sp. PI12）, and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene （named JSP8A） showing 60% identity （98% query coverage） to subtilisin peptidases belonging to the $8 family （S8A subfamily） of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from SSA bacterial sequences of other phyla （including its own）. An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer （HGT） from a cyanobacterittrn. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis.

Evolution of PE35 and PPE68 Gene Families in Mycobacterium: Roles of Horizontal Gene Transfer and Evolutionary Constraints

Mycobacterium is a genus of bacteria with over a hundred non-pathogenic and pathogenic species, best recognized for certain members known to cause diseases such as tuberculosis and leprosy. Two novel protein families important in the pathogenesis of Mycobacterium species are the PE and PPE families. These two protein families affect the antigenic profiles, disturbing host immunity. To better understand the origin and evolution of these gene families and the differences in their composition between pathogenic and non-pathogenic strains, several bioinformatic analyses were conducted both among Mycobacterium and closely related species that contain PE35 and PPE68 gene homologs. The methods included protein homology searches (BLASTP), horizontal gene transfer analysis (IslandViewer), phylogenetic analysis, gene cluster analysis and structural and functional constraints. Results revealed that PE and PPE gene homologs were not only limited to Mycobacterium, but also existed in three other non-mycobacterial genera, Rhodococcus, Tsukamurella and Segniliparus, and were possibly initially acquired from non-mycobacterial microorganisms by multiple horizontal gene transfers. Results also demonstrated that PE and PPE genes were more diverse and more rapidly evolving in pathogenic Mycobacterium as compared with non-pathogenic Mycobacterium and other non-mycobacterial species. These findings possibly shed light on the diverse functions and origins of the PE/PPE proteins among these organisms.

Prokaryotic Horizontal Gene Transfer in Freshwater Lakes: Implications of Dynamic Biogeochemical Zonation

The highly adaptive nature of prokaryotic communities in the face of changing environmental conditions reflects in part their ability to share advantageous genetic information through horizontal gene transfer (HGT). Natural freshwater lacustrine (lake) systems are a vital and finite resource, and the influence of HGT on their quality (e.g. enabling the spread of antibiotic resistance and xenobiotic catabolism genes) is likely significant. Laboratory and in situ studies indicate that the dynamic physical, chemical, and biological conditions that structure freshwater systems can influence HGT within freshwater prokaryotic communities. Thus, understanding how biogeochemical parameters impact HGT in freshwater lakes is an emerging knowledge gap with potential implications for ecosystem and human health on a global scale. In this review, we provide a general synopsis of what is known about HGT in freshwater prokaryotic communities, followed by an integrated summary of current knowledge identifying how biogeochemical factors may influence prokaryotic HGT in freshwater lacustrine systems.

An acyltransferase gene that putatively functions in anthocyanin modification was horizontally transferred from Fabaceae into the genus Cuscuta

Horizontal gene transfer(HGT) refers to the flow of genetic materials to non-offspring,and occasionally HGT in plants can improve the adaptation of organisms in new niches due to expanded metabolic capability.Anthocyanins are an important group of water-soluble red,purple,or blue secondary metabolites,whose diversity results from modification after the main skeleton biosynthesis.Cuscuta is a stem holoparasitic genus,whose members form direct connection with hosts to withdraw water,nutrients,and macromolecules.Such intimate association is thought to increase the frequency of HGT.By transcriptome screening for foreign genes in Cuscuta australis,we discovered that one gene encoding a putative anthocyanin acyltransferase gene of the BAHD family,which is likely to be involved in anthocyanin modification,was acquired by C.australis from Fabaceae through HGT.The anthocyanin acyltransferase-like(AT-like) gene was confirmed to be present in the genome assembly of C.australis and the transcriptomes of Cuscuta pentagona.The higher transcriptional level in old stems is consistent with its putative function in secondary metabolism by stabilizing anthocyanin at neutral pH and thus HGT of this AT-like gene may have improved biotic and abiotic resistance of Cuscuta.

Horizontal gene transfer of a syp homolog contributes to the virulence of Burkholderia glumae

Horizontal gene transfer(HGT)has been proved a major driving force in prokaryotic evolution.However,the molecular functions of these transferred genes in pathogenic bacteria especially plant pathogenic bacteria are still not fully investigated.In this study,the whole-genome in silico analysis was performed and found a syringopeptin synthetase(syp)homolog in Burkholderia glumae,which can cause bacterial panicle blight in rice,was predicted to be horizontally transferred from Pseudomonas ancestor with solid confidence by phylogenetic analysis.The comprehensive molecular experiments were performed to study the potential role of this gene in B.glumae.Inoculation of rice panicles with the syp mutant resulted in 60%lower disease index compared with the wild type(WT)parent strain,suggesting the requirement of syp for the full virulence of B.glumae.Chromatography analysis of exudates from B.glumae showed suppression of synthesis of metabolites analogous to syringopeptin in the mutants.All these data raise the possibility of HGT phenomenon in shaping the virulence and adaptation of B.glumae over evolutionary time.

Progress in gene transfer by germ cells in mammals

Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences. Such approach is referred to as either male germ cell mediated gene transfer （MGCMGT） or female germ cell mediated gene transfer （FGCMGT） technique. Sperm-mediated gene transfer （SMGT）, including its alternative method, testis-mediated gene transfer （TMGT）, becomes an established and reliable method for transgenesis. They have been extensively used for producing transgenic animals. The newly developed approach of FGCMGT, ovary-mediated gene transfer （OMGT） is also a novel and useful tool for efficient transgenesis. This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques, methods developed and mechanisms of nucleic acid uptake by germ cells.

Unexpected complex horizontal gene transfer in teleost fish

Horizontal gene transfer(HGT)is a common occurrence across all domains of life.However,most HGT events were reported between single-celled organisms or parasites and hosts(Van Etten and Bhattacharya 2020).A type II antifreeze protein(AFP)gene was the first and sole evidence of HGT direct vertebrate-to-vertebrate DNA transmission.AFP is only found in 3 widely separated branches of teleost fishes(herring,sea raven,and smelts),sharing amino acid similarity up to 80%(Graham et al.2008).

Fluid Flow and Mixed Heat Transfer in a Horizontal Channel with an Open Cavity and Wavy Wall

Heat exchangers are utilized extensively in different industries and technologies.Consequently,optimizing heat exchangers has been a major concern among researchers.Although various studies have been conducted to improve the heat transfer rate,the use of a wavy wall in the presence of different types of heat transfer mechanisms has not been investigated.This study thus investigates the mixed heat transmission behavior of fluid in a horizontal channel with a cavity and a hot,wavy wall.The fluid flow in the channel is considered laminar,and the governing equations including continuity,momentum,and energy are all solved numerically.The numerical solution is stabilized by using a first-order multi-dimensional characteristic-based scheme in combination with a fifth-order Runge-Kutta method.The flow and heat transfer effects of varying Richardson numbers,Reynolds numbers,wave amplitude,wavelength,channel height,and cavity width are examined.The results indicate that the mean Nusselt number increases with an increase in Reynolds number,wave amplitude,and cavity width,while it decreases with an increase in Richardson number,wavelength,and channel height.The minimum Nusselt number is calculated to be 0.7,whereas the maximum Nusselt number is 27.09.The Nusselt number has only increased by 40%in the higher depths of the cavity,despite the Richardson number being 10,000 times larger.But this figure increases to 130%at lower depths.The mean Nusselt number is thus significantly influenced by channel height and cavity width.The influence of wave amplitude on the mean Nusselt number is twice that of wavelength.

Testing the possibility of horizontal transfer of introduced neomycin phosphotransferase(nptII) gene of transgenic Eucalyptus camaldulensis into soil bacteria

The possible horizomal transfer of transgenes is of great concern when the transgenic plants are released imo the field. To test the possible transfer of nptII of transgenic trees into soil bacteria, we have used a stool DNA preparation kit to isolate the DNA from the soils in the rhizospheres of two non- and eight transgenic Eucalyptus camaldulensis trees. All the samples have provided the corresponding PCR products in the amplification with bacterial 16S RNA specific sequences, which indicates that the quality of the isolated DNA is adequate for amplification. The nptⅡ specific band has been amplified in three soil samples from the transgenic trees and even treated with filtration before the DNA isolation. This indicates that nptII DNA exists in the soil, although it is still unclear whether the DNA was in the soil particles, in the soil bacteria or in the Agrobacterium comamination which was used for the E. camaldulensis transformation. Two approaches on isolation of bacterial DNA have been suggested for testing the possibility of this event in the future.

cGH Gene Transfer and PCR Detection in Crucian Carp

By inserting the CMV with HSV TK promoter into the plasmid pBlue-cGH which contained cGH gene, a chimeric expression vector was constructed for transgenic fish. After the vector was transferred into fertilized eggs of crucian carp by means of microinjection, the transgenic crucian carp was obtained. The result of PCR detection showed that the growth hormone gene of the exogenous carp was integrated into the genome of crucian carp, and the transgenic crucian carp displayed enhancement of its growth.

Horizontal gene transfer of plant-specific leucine-rich repeats between plants and bacteria

Leucine rich repeats (LRRs) are present in over 14,000 proteins that have been identified in viruses, bacteria, archaea, and eukaryotes. Two to sixty-two LRRs occur in tandem forming an overall arc shaped domain. There are eight classes of LRRs. Plant specific LRRs (class: PS-LRR) had previously been recognized in only plant proteins. However, we find that PS-LRRs are also present in proteins from bacteria. We investigated the origin of bacterial PS-LRR domains. PSLRR proteins are widely distributed in most plants;they are found in only a few bacterial species. There are no PS-LRR proteins from archaea. Bacterial PS-LRRs in twenty proteins from eleven bacterial species (in the three phyla: Proteobacteria, Cyanobacteria, and Bacteroidetes) are significantly more similar to the PS-LRR class than to the other seven classes of LRR proteins. Not only amino acid sequences but also nucleotide sequences of the bacterial PS-LRR domains show highly significant similarity with those of many plant proteins. The program, EGID (Ensemble algorithm for Genomic Island Detection), predicts that Synechococcus sp. CYA_ 1022 came from another organism. Four bacterial PS-LRR proteins contain AhpC-TSA, IgA peptidase M64, the immunoglobulin domain, the Calx-b domain, and the He_PIG domain;these domains show no similarity with any eukaryotic (plant) proteins, in contrast to the similarities of their respective PS-LRRs. The present results indicate that horizontal gene transfer (HGT) of genes/gene fragments encoding PS-LRR domains occurred between bacteria and plants, and HGT among the eleven bacterial species, of the three phyla, as opposed to descent from a common ancestor. There is the possibility of the occurrence of one HGT event from plant to bacteria. A series of HGTs might then have occurred recently and rapidly among these eleven species of bacteria.

Adenoviral-mediated Hath1-EGFP gene transfer into guinea pig cochlea through intact round window membrane

Objective To study expression of adenoviral-mediated Hath1-EGFP gene in the guinea pig cochlea after transfer through intact round window membrane(RWM), and to assess its effects on hearing. Methods Twenty adult guinea pigs were used, of which: 12 were surgically inoculated with Ad-Hath1-EGFP in the bony groove of round window niche, and 8 with artificial perilymph. Auditory brainstem response(ABR) thresholds were determined in all animals before and 5 days after surgery. On post-surgery day 5 and day 14, animals were sacrificed and whole mounts of cochlea and frozen sections were examined. Results ABR tests showed no significant change of hearing after the surgery. Strong fluorescence staining in the cochleae was seen in Ad-Hath1-EGFP groups. The highest levels of gene expression were seen in the post-surgery day 5 group with little decrease on post-surgery day 14.The contralateral cochlea and those in the control groups were free of fluorescence staining. Conclusion The transgenic Hath1-EGFP can be effectively delivered into the inner ear through intact RWM, in an atraumatic manner.

Adenovirus-mediated FasL gene transfer into human gastric carcinoma

AIM: To evaluate the possible value of FasL in gastric cancer gene therapy by investigating the effects of FasL expression on human gastric cancer cell line. METHODS: An adenoviral vector encoding the full-length human FasL cDNA was constructed and used to infect a human gastric cancer (SGC-7901) cell line. FasL expression was confirmed by X-gal staining, flow cytometric analysis and RT-PCR. The effect of FasL on cell proliferation was determined by clonogenic assay, cytotoxicity was detected by MTT assay, and cell viability was measured by trypan blue exclusion. The therapeutic efficiency of Ad-FasL in vivo was investigated with a xenograft tumor model in nude mice. RESULTS: SGC-7901 cells infected with Ad-FasL showed increased expression of FasL, resulting in significantly decreased cell growth and colony-forming activity when compared with control adenovirus-infected cells. The cytotoxicity of anti-Fas antibody (CH-11) in gastric cancer cells was stronger than that of ActD (91±8 vs60±5,P<0.01), and the cytotoxicity of Ad-FasL was stronger than that of CH-11 (60±5 vs50±2, P<0.05). In addition, G1-phase arrest (67.75±0.39 vs 58.03±2.16, P<0.05) and apoptosis were observed in Ad-FasL-infected SGC-7901 cells, and the growth of SGC-7901 xenografts in nude mice was retarded after intra-tumoral injection with Ad-FasL (54% vs 0%,P<0.0001). CONCLUSION: Infection of human gastric carcinoma cells with Ad-FasL induces apoptosis, indicating that this target gene might be of potential value in gene therapy for gastric cancer.

Microbubble-enhanced ultrasound exposure improves gene transfer in vascular endothelial cells

AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluorescent protein （pEGFP） transfer into human umbilical vein endothelial cells （HUVECs）. METHODS： HUVECs with fluorescein isothiocyanatedextran （FD500） and HUVECs with pEGFP were exposed to continuous wave （1.9 MHz, 80.0 mW/cm^2） for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytornetry, respectively. RESULTS： The percentage of FDS00-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone （24.0%± 5.5% vs 66.6% ± 4.1%, P 〈 0.001）. Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue （16.1% ± 1.9% vs 1.5% ± 0.2%, P 〈 0.001）. No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue （94.1% ± 2.3% vs 91.1% ± 4.1% ）. CONCLUSION： The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non- invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.

Erythropoietin gene transfer into rat testes by in vivo electropo-ration may reduce the risk of germ cell loss caused by cryptorchidism

Aim： To investigate the effects of rat Erythropoietin （Epo） on spermatogenesis by transferring rat Epo gene into cryptorchid testes by means of in vivo electroporation. Methods： Sprague-Dawley rats with surgically-induced unilateral cryptorchidism were divided into three groups： the first group was given intratesticular injections of pCAGGS-Epo （pCAGGS-Epo group）, the second group was given intratesticular injections of pCAGGS （pCAGGS group）, and the third group were given intratesticular injections of phosphate-buffered saline （PBS group）. At the same time, square electric pulses of 30 V were applied six times with a time constant of 100 ms. One or two weeks after injection, each testis was weighed and the ratio of the total number of germ cells to that of Sertoli cells （G/S ratio） was calculated to evaluate the impairment of spermatogenesis. Ten testes taken from each of the three groups were examined at each time point. Results： The testicular weight after the injection of pCAGGS-Epo or pCAGGS control plasmid was （0.85 ± 0.08） g and （0.83 ± 0.03） g, respectively, at week 1 （P = 0.788） and （0.62 ± 0.06） g and （0.52 ± 0.02） g, respectively, at week 2 （P = 0.047）. At week 1, spermatids and sperm were more abundant in testes with pCAGGS-Epo than those in the control testes. At week 2, spermatids and sperm were hardly detected in either group. The G/S ratio was 23.27 ± 6.80 vs. 18.63 ± 5.30 at week 1 （P = 0.0078） and 7.16 ± 3.06 vs. 6.05 ± 1.58 at week 2 （P = 0.1471）, respectively. Conclusion： The transfer of Epo to rat testes by in vivo electroporation may reduce the risk of the germ cell loss caused by cryptorchidism.

Numerical investigation of laminar heat transfer and nanofluid flow between two porous horizontal concentric cylinders

In this study, the laminar heat transfer and nanofluid flow between two porous horizontal concentric cylinders was investigated. The problem is investigated in two different geometries and the Re=10, 25, 50, 75, 100 and volume fraction 0, 0.2%, 0.5%, 2% and 5% that related to copper nanoparticles, and porous medium porosity of 0.5 and 0.9. Compared to the first geometry, the convective coefficient in the second geometry increases by 8.3%, 7% and 5.5% at Reynolds numbers of 100, 75 and 50, respectively. Comparison of the outlet temperatures for two heat fluxes of 300 and 1200 W/m^2 indicates a 2.5% temperature growth by a fourfold increase in the heat fluxes. Also, the higher Nusselt number is associated with the second geometry occurring at porosities of 0.9 and 0.5, respectively. In both geometries, the Nusselt number values at the porosity of 0.9 are higher, which is due to the increased nanofluid convection at higher porosities. The velocity of the nanofluid experiences a two-fold increase at the outlet compared to its inlet velocity in the first geometry and for both porosities. Similarly, a three-fold increase was achieved in the second geometry and for both porosities.

Intrastriatal Gene Transfer of Vascular Endothelial Growth Factor Rescues Dopaminergic Neurons in a Rat Parkinson's Disease Model

To examine the ability of intrastriatal gene transfer of vascular endothelial growth factor 165 mediated by adenoviral vector to rescue dopaminergic neurons in a rat model of Parkinson＇s disease （PD）, we constructed recombinant replication-deficent adenoviral vectors carrying the gene of VEGF165 （Ad-VEGF）, and injected Ad-VEGF （or Ad-LacZ and PBS as controls） into the striatum of rats 7 days after the lesion by 6-hydroxydopamine. The rat rotational behavior analysis and tyrosine hydroxylase （TH） immunohistochemistry were performed to assess the change of dopaminergic neurons. Our results showed that the rats receiving Ad-VEGF injection displayed a significant improvement in apomorphine-induced rotational behavior and a significant preservation of TH-positive neurons and fibers compared with control animals, It is concluded that intrastriatal gene transfer by Ad-VEGF may rescue the dopaminergic neurons from degeneration in a rat model of PD.

Molecular Tissue Engineering: Applications for Modulation of Mesenchymal Stem Cells Proliferation by Transforming Growth Factor β_1 Gene Transfer

The effect of transforming growth factor β 1 (TGF β 1 ) gene transfection on the proliferation of bone marrow derived mesenchymal stem cells (MSC S ) and the mechanism was investigated to provide basis for accelerating articular cartilage repairing using molecular tissue engineering technology. TGF β 1 gene at different doses was transduced into the rat bone marrow derived MSCs to examine the effects of TGF β 1 gene transfection on MSCs DNA synthesis, cell cycle kinetics and the expression of proliferating cell nuclear antigen (PCNA). The results showed that 3 μl lipofectamine mediated 1 μg TGF β 1 gene transfection could effectively promote the proliferation of MSCs best; Under this condition (DNA/Lipofectamine=1μg/3μl), flow cytometry and immunohistochemical analyses revealed a significant increase in the 3 H incorporation, DNA content in S phase and the expression of PCNA. Transfection of gene encoding TGF β 1 could induce the cells at G0/G1 phase to S1 phase, modulate the replication of DNA through the enhancement of the PCNA expression, increase the content of DNA at S1 phase and promote the proliferation of MSCs. This new molecular tissue engineering approach could be of potential benefit to enhance the repair of damaged articular cartilage, especially those caused by degenerative joint diseases.

Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture

AIM. To investigate the influence of heme oxygenase-1 （HO-1） gene transfer on the viability and function of cultured rat islets in vitro. METHODS： Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene （Ad- HO-1） or enhanced green fluorescent protein gene （Ad- EGFP）, and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/ propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index （SI） was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS： After seven days culture, the viability of cultured rat islets decreased significantly （92% ± 6% vs 52% ± 13%, P 〈 0.05）, and glucose-stimulated insulin release also decreased significantly （6.47 ± 0.55 mIU/ L/30IEO vs 4.57 ± 0.40 mIU/L/3OIEO., 14.93 ± 1.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. Transfection of rat islets with adenoviral vectors at an 1±10 of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets（71% ± 15% vs 52% ± 13%, P 〈 0.05）. There was no significant difference in insulin release upon low glucose stimulation （2.8 mmol/L） among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group （P 〉 0.05）, while when stimulated by high glucose （16.7 mmol/L） solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively （12.50 ±2.17 mIU/L/30IEQ vs 8.87 ± 0.65 mIU/L/30IEQ, 12.50 ± 2.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively （2.21 ± 0.02 vs 2.08 ± 0.05; 2.21 ± 0.02 vs 2.11 ± 0.03, P 〈 0.05）. CONCLUSION： The viability and function of rat islets decrease over time in in vitro culture, and heine oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.

HIGH EFFICIENCY RETROVIRUS-MEDIATED GENE TRANSFERTO LEUKEMIA CELLS

Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1. The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107 CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR). Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days’ culture. Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.