Current studies have shown that centromere protein F (CENPF) was overexpressed in hepatocellular carcinoma (HCC) and might be involved in the pathogenesis of HCC. Specifically, due to the very large molecular weight (358 kDa) of CENPF full length protein, only CENPF knock-down, but not overexpression models, were applied currently to explore the carcinogenicity of CENPF in HCC. Whether CENPF overexpression is a cause or an effect in HCC remains to be illustrated. We aimed to establish a CENPF overexpression cell model using CRISPR/dCas9 synergistic activation mediator (SAM) system with lentiMPHv2 and lentiSAMv2 vectors to explore the role of CENPF overexpression in HCC. Single guide RNAs (sgRNAs) that specifically identify the transcription initiation site of CENPF gene were synthesized and inserted into the lentiSAMv2 plasmid. Huh-7 and HCCLM3 cells were first transduced with lentiMPHv2 and then selected with hygromycin B. The cells were then transduced with lentiSAMv2 carrying specific sgRNA for CENPF gene, followed by blasticidin S selection. The mRNA and protein detection results of Huh-7 and HCCLM3 cells screened by hygromycin B and blasticidin S showed that the endogenous overexpression of CENPF can be induced by sgRNA1 and sgRNA4, especially by sgRNA4. By using the CRISPR/dCas9 technique, stable cell models with overexpressed CENPF were successfully constructed to explore the role of CENPF in tumorigenesis, which provides a reference for the construction of cell models overexpressing large molecular weight protein.
Humans ; Carcinoma, Hepatocellular/genetics* ; Liver Neoplasms/genetics* ; RNA, Guide, CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Hygromycin B

To construct a system of genetic transformation suitable for Rhizopus oryzae, we constructed a single-exchange vector pBS-hygro carrying hygromycin B resistance gene (hph) as its selective marker using gene splicing by overlap extension PCR (SOE PCR) technique. We introduced this recombinant vector into Rhizopus oryzae AS 3.819 by PEG/CaCl2-mediated transformation of protoplast, electroporation of protoplast and germinated spores; and we studied the effects of hydrolysis time, field strength and spore germination time on transformation frequency. We conducted quantitative real-time PCR (qPCR) assay to determine the gene copy number of ldhA integrated in the genome of R. oryzae transformants and its effect on the stability of transformants. We successfully achieved R. oryzae transformants integrated with pBS-hygro-ldhA vector. The optimal hydrolysis time for protoplast production was 140 min, and the optimal field strength of electroporation pulse for protoplast was 13 kV/cm. The optimal germination time of spores for electroporation was 2.5 h, and the optimal field strength of electroporation pulse was 14 kV/cm. The transformation frequency of method based on germinated spores was generally higher than the methods based on protoplast. The qPCR test results suggested that transformants with high copy number of integration in a certain range were relatively stable. Our results provided basis and support for metabolic regulation and genetic engineering breeding of R. oryzae.
DNA, Recombinant ; Electroporation ; Genetic Engineering ; Genetic Vectors ; Hygromycin B ; Protoplasts ; Real-Time Polymerase Chain Reaction ; Rhizopus ; genetics ; Transformation, Genetic

We developed pancreatic and duodenal homeobox1 (Pdx1) knockout mice to improve a compensatory hyperinsulinemia, which was induced by hyperplasia in the beta cells or Langerhans' islands, as the diabetic model mice. For targeting of Pdx1 gene by homologous recombination, ES cells derived from a 129(+Ter)/SvJclxC57BL/6JJcl hybrid mouse were electroporated and subjected to positive-negative selection with hygromycin B and ganciclovir. As these results, one of the three chimeric mice succeeded to produce the next or F1 generation. Then, the mouse fetuses were extracted from the mother's uterus and analyzed immunohistologically for the existence of a pancreas. The fetuses were analyzed at embryonic day 14.5 (E14.5) because Pdx1 knockout could not alive after birth in this study. Immunohistochemical staining revealed that 10 fetuses out of 26 did not have any PDX1 positive primordium of the pancreas and that the PDX1 expresses in both the interior and exterior regions of intestine. In particular, one the exterior of the intestine PDX1 was expressed in glands that would be expected to form the pancreas. The result of PCR genotyping with extracted DNA from the paraffin sections showed existence of 10 Pdx1-knockout mice and corresponded to results of immunostaining. Thus, we succeeded to establish a Pdx1-knockout (Pdx1-/-) mice.
Animals ; DNA ; Fetus ; Ganciclovir ; Homologous Recombination ; Hygromycin B ; Hyperinsulinism ; Hyperplasia ; Intestines* ; Islands ; Mice ; Mice, Knockout* ; Pancreas ; Paraffin ; Parturition ; Polymerase Chain Reaction ; Uterus

<b>OBJECTIVEb>To construct plant expression pCAMBIA1301-PMI by substituting PMI for hygromycin resistance gene in pCAMBIA1301 and obtain transgenic Salvia miltiorrhiza f. alba using PMI-mannose selection system.

<b>METHODb>The 6-phosphomannose isomerase gene (PMI) of Escherichia coli was amplified by PCR. Sequence analysis showed that it shared 100% amino acids identities with the sequences of PMI genes isolates reported in the NCBI. Based on pCAMBIA1305, the plant expression pCAMBIA1305-PMI was constructed successfully by substituting PMI for hygromycin resistance gene in pCAMBIA1305. pCAMBIA1305-PMI was transformed into Agrobacterium tumefaciens LBA4404, and then the leaves of S. miltiorrhiza f. alba were inoculated in LBA4404 with pCAMBIA1305-PMI.

<b>RESULTb>Plant expression pCAMBIA1301-PMI was successfully constructed and the leaves of S. miltiorrhiza f. alba inoculated in LBA4404 with pCAMBIA1305-PMI were selected on medium supplemented with a combination of 20 g x L(-1) mannose and 10 g x L(-1) sucrose as a carbon source. The transformation efficiency rate was 23.7%.

<b>CONCLUSIONb>Genetic transformation was confirmed by PCR, indicating that a new method for obtaining transgenic S. miltiorrhiza f. alba plants was developed using PMI-mannose selection system.

Anti-Bacterial Agents ; pharmacology ; Biomarkers ; Cinnamates ; pharmacology ; Escherichia coli ; enzymology ; genetics ; Escherichia coli Proteins ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Hygromycin B ; analogs & derivatives ; pharmacology ; Mannose-6-Phosphate Isomerase ; genetics ; metabolism ; Plants, Genetically Modified ; drug effects ; genetics ; metabolism ; Salvia miltiorrhiza ; drug effects ; genetics ; metabolism ; Transformation, Genetic

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/microg of DNA in 1 x 107 protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.
Agaricales ; Blotting, Southern ; Chimera ; Cinnamates ; Coat Protein Complex I ; DNA ; Electroporation ; Flammulina ; Fungal Proteins ; Genome ; Hygromycin B ; Mycotoxins ; Oxidoreductases ; Plasmids ; Polymerase Chain Reaction ; Protoplasts ; Recombination, Genetic ; Sprains and Strains

Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 10(6) conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.
Agrobacterium tumefaciens ; genetics ; Base Sequence ; Citrus ; microbiology ; DNA Primers ; genetics ; DNA, Bacterial ; genetics ; DNA, Fungal ; genetics ; DNA, Recombinant ; genetics ; Drug Resistance, Fungal ; genetics ; Hygromycin B ; pharmacology ; Molecular Sequence Data ; Mutagenesis, Insertional ; Penicillium ; drug effects ; genetics ; pathogenicity ; Plant Diseases ; microbiology ; Plasmids ; genetics ; Transformation, Genetic

A strain Mortierella isabellina M6-22-4, which was sensitive to hygromycin B, was selected by treating parental spores with N-methyl-N' -Nitro-N-nitrosoguanidine (MNNG). Protoplasts of the strain Mortierella isabellina M6-22-4 were transformed successfully to hygromycin B resistance using the PD4 plasmid, which contains the Escherichia coli hph gene under the control of Mortierella alpina his H4.1 promoter. The PD4 plasmid was introduced by PEG/CaCl2 treatment. Transformation frequencies of 1.6 - 2.8 transformants/microg of DNA were achieved. Then they were successively incubated to non-selected PDA plates for 10 generations. About 31.6% transformants only from digested plasmid were mitotically stable and showed different hygromycin B resistance when they were incubated back to selection plates. The results of PCR and Southern analysis in three transformants indicated that the plasmid PD4 had been integrated into the fungal genome with 1 - 2 copies. This is the first report of Mortierella isabellina transformation system and supplies an important tool for further research into genetic manipulation of this filamentous fungus.
Anti-Bacterial Agents ; pharmacology ; Blotting, Southern ; Drug Resistance, Microbial ; genetics ; Escherichia coli Proteins ; genetics ; Genome, Fungal ; genetics ; Hygromycin B ; pharmacology ; Mortierella ; drug effects ; genetics ; growth & development ; Plasmids ; genetics ; Polymerase Chain Reaction ; Protoplasts ; metabolism ; Transformation, Genetic

To generate transgenic mice in which both hygromycin (hyg) and neomycin (neo) resistance genes are expressed in murine fibroblast cells (MEFs), which are required for conditional gene knock-out and screening of drug resistant ES cell clones. To construct HygR-neoR expression vector, pTK-hygR-pA and PGK-neoR-pA were cloned into pBluescript vector. DNA fragments of tandem genes ( 4245bp ) were prepared by Kpn I and Xba I digestion and transgene was microinjected into pronucleus of zygotes to generate transgenic mice. Transgenic mice were identified by PCR and Southern blot; expression of hygR and neoR gene transcripts were detected by RT-PCR. 7 founder mice carrying hyg-neo resistant genes were obtained and 6 transgenic mouse lines were successfully established. The hygR and neoR gene transcripts were detected in the liver and/or ovary of transgenic mice from hn30, hn33, hn66 and hn67 mouse lines. In MEFs isolated from the mice of line hn66 and hn30, expression of hyg and neo resistant genes was also detectable. Transgenic mouse lines expressing two anti-drug genes have been established. The hyg and neo resistant gene transcripts were detected in the MEFs of two transgenic mouse lines.
Animals ; Cinnamates ; pharmacology ; Drug Resistance, Multiple ; genetics ; Fibroblasts ; metabolism ; Hygromycin B ; analogs & derivatives ; pharmacology ; Mice ; Mice, Transgenic ; Neomycin ; pharmacology ; Transgenes ; genetics

<b>OBJECTIVEb>To cooperate with the study of HBV vector, hygromycin-resistant packaging cell line was developed that allows encapsidation of plasmids into HBV particles.

<b>METHODSb>Free of packaging signal, HBV genome was inserted into plasmid pMEP4, which expresses the HBV structural proteins including core, pol and preS/S proteins. HepG2 cell lines were employed to transfect with the construct. Hygromycin selection was done at a concentration of 150 micrograms/ml in the culture medium. The hygromycin-resistant clones with the best expressions of HBsAg and HBcAg were theoretically considered as packaging cell line and propagated under the same conditions. It was infected with recombinant retrovirus vector and hen selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR.

<b>RESULTSb>Hygromycin-resistant HBV packaging cell line was generated, which harbored an HBV mutant whose packaging signal had been deleted. Expressions of HBsAg and HBcAg were detectable. Infected with recombinant retrovirus pRV-CP, the hygromycin-resistant packaging cell line was found to secrete mutant HBV particles and no wild-type HBV was detectable in the culture medium.

<b>CONCLUSIONb>After the packaging signal was deleted and transfected into HepG2 cell lines, the partial HBV genome lost its ability to form wild-type HBV, but conserves cis-action providing structural proteins for the packaging of the replication-defective HBV.

Cell Line ; Drug Resistance, Viral ; Genetic Vectors ; Genome, Viral ; Hepatitis B virus ; drug effects ; genetics ; Humans ; Hygromycin B ; pharmacology ; Mutation ; Plasmids ; Retroviridae ; genetics ; Transfection ; Virus Assembly

The rice blast fungus Magnaporthe grisea causes one of the most destructive diseases of rice around the world. Significant progresses have been made recently in genomics studies of the fungus, opening new era of the functional genomics which requires to generate a large scale of gene knockout mutants. It has been demonstrated that T-DNA insertional mutagenesis is a powerful tool of functional genomics not only for plants but also for fungi. In this paper, we optimized the conditions for T-DNA insertional mutagenesis of M. grisea using Agrobacterium tumefaciens-mediated transformation (ATMT) approach. We employed the binary vector pBHtl constructed by Dr. S. Kang's laboratory at the Pennsylvania State University, which carries the bacterial hygromycin B phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker to transform the conidia of M. grisea. We optimized the conditions for T-DNA insertional mutagenesis including the medium, dosage of hygromycin B, cefotaxime and carbenicillin to select the transformants and inhibit the growth of A. tumefaciens after co-culturing. The dosage to inhibit non-transformants could vary from 200-600microg/mL among different M. grisea isolates so that the optimal dosage of the antibiotics should be decided according to isolates. Rice polished agar medium was found the best selection medium which would facilitate the mutant sporulation and minimize the contamination chance. In average, about 500 transformants could be obtained when transforming 1 x 10(6) spores at the optimum condition, among which 85% had T-DNA insertion detected by polymerase chain reaction (PCR) and thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). Fifteen out of 1520 transformants showed mutation in colony morphology. Within 58 randomly selected mutants, it was found that there were 4 sporulation-decreased mutants, 8 less germination mutants and 9 appressorium defective mutants. Several virulent mutants to C101LAC(Pi-1)and 75-1-127(Pi-9)were also obtained which would facilitate cloning the corresponding avirulence genes.
Agrobacterium tumefaciens ; genetics ; Bacterial Proteins ; genetics ; Carbenicillin ; pharmacology ; Cefotaxime ; pharmacology ; DNA, Bacterial ; genetics ; physiology ; Genetic Vectors ; genetics ; Hygromycin B ; pharmacology ; Magnaporthe ; drug effects ; genetics ; Mutagenesis, Insertional ; methods ; Mutation ; Oryza ; microbiology ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Transformation, Genetic ; genetics ; physiology