scholarly journals Rapid, high efficiency virus-mediated mutant complementation and gene silencing in Antirrhinum

Plant Methods ◽  
2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Ying Tan ◽  
Alfredas Bukys ◽  
Attila Molnár ◽  
Andrew Hudson
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
High Efficiency ◽  
Tobacco Rattle Virus ◽  
Transient Gene Expression ◽  
Mutant Phenotype ◽  
Homologous Sequence ◽  
Homologous Gene ◽  
Loss Of Function ◽  
H Protein

Abstract Background Antirrhinum (snapdragon) species are models for genetic and evolutionary research but recalcitrant to genetic transformation, limiting use of transgenic methods for functional genomics. Transient gene expression from viral vectors and virus-induced gene silencing (VIGS) offer transformation-free alternatives. Here we investigate the utility of Tobacco rattle virus (TRV) for homologous gene expression in Antirrhinum and VIGS in Antirrhinum and its relative Misopates. Results A. majus proved highly susceptible to systemic TRV infection. TRV carrying part of the Phytoene Desaturase (PDS) gene triggered efficient PDS silencing, visible as tissue bleaching, providing a reporter for the extent and location of VIGS. VIGS was initiated most frequently in young seedlings, persisted into inflorescences and flowers and was not significantly affected by the orientation of the homologous sequence within the TRV genome. Its utility was further demonstrated by reducing expression of two developmental regulators that act either in the protoderm of young leaf primordia or in developing flowers. The effects of co-silencing PDS and the trichome-suppressing Hairy (H) gene from the same TRV genome showed that tissue bleaching provides a useful marker for VIGS of a second target gene acting in a different cell layer. The ability of TRV-encoded H protein to complement the h mutant phenotype was also tested. TRV carrying the native H coding sequence with PDS to report infection failed to complement h mutations and triggered VIGS of H in wild-type plants. However, a sequence with 43% synonymous substitutions encoding H protein, was able to complement the h mutant phenotype when expressed without a PDS VIGS reporter. Conclusions We demonstrate an effective method for VIGS in the model genus Antirrhinum and its relative Misopates that works in vegetative and reproductive tissues. We also show that TRV can be used for complementation of a loss-of-function mutation in Antirrhinum. These methods make rapid tests of gene function possible in these species, which are difficult to transform genetically, and opens up the possibility of using additional cell biological and biochemical techniques that depend on transgene expression.

Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency

1982 ◽  
Vol 2 (9) ◽  
pp. 1145-1154
Author(s):  
Y M Shen ◽  
R R Hirschhorn ◽  
W E Mercer ◽  
E Surmacz ◽  
Y Tsutsui ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Thymidine Kinase ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Transient Gene Expression ◽  
Simian Virus 40 ◽  
Temperature Sensitive ◽  
Enzymatic Function ◽  
Very High

We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (approximately 10(4) base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk-ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.

esiRNAs Purified With Chromatography Suppress Homologous Gene Expression With High Efficiency and Specificity

2005 ◽  
Vol 31 (3) ◽  
pp. 203-210 ◽  
Author(s):  
Baoqin Xuan ◽  
Zhikang Qian ◽  
Chang Tan ◽  
Taishan Min ◽  
Shuiyuan Shen ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Efficiency ◽  
Homologous Gene

scholarly journals An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves

Rice ◽  
2012 ◽  
Vol 5 (1) ◽  
pp. 23 ◽  
Author(s):  
Aurélie Andrieu ◽  
Jean Breitler ◽  
Christelle Siré ◽  
Donaldo Meynard ◽  
Pascal Gantet ◽  
...  
Keyword(s):  
Gene Expression ◽  
Oryza Sativa ◽  
Gene Silencing ◽  
Oryza Sativa L ◽  
Transient Gene Expression ◽  
In Planta

scholarly journals Virus-Induced Gene Silencing and Transient Gene Expression in Soybean (Glycine max) Using Bean Pod Mottle Virus Infectious Clones

2016 ◽  
Vol 1 (2) ◽  
pp. 263-283 ◽  
Author(s):  
Steven A. Whitham ◽  
Lori M. Lincoln ◽  
R. V. Chowda-Reddy ◽  
Jaime D. Dittman ◽  
Jamie A. O'Rourke ◽  
...  
Keyword(s):  
Gene Expression ◽  
Glycine Max ◽  
Gene Silencing ◽  
Transient Gene Expression ◽  
Mottle Virus ◽  
Virus Induced Gene Silencing ◽  
Bean Pod Mottle Virus ◽  
Infectious Clones

scholarly journals Development of an Efficient Transient Gene Expression Assay Based on Tobacco ((Nicotiana tabacum var. Xanthi) Male Gametophytes

1970 ◽  
Vol 19 (1) ◽  
pp. 9-23 ◽  
Author(s):  
M. A. Y. Akhond ◽  
G. C. Machray
Keyword(s):  
Gene Expression ◽  
Nicotiana Tabacum ◽  
Expression System ◽  
Transient Gene Expression ◽  
Specific Gene ◽  
Loss Of Function ◽  
Pollen Selection ◽  
Gene Expression Assay ◽  
Study Gene Expression

Optimization of direct DNA delivery into tobacco ((Nicotiana tabacum var. Xanthi) male gametophytes was devised together with development of an efficient transient expression system to study gene expression under controlled conditions. Use of a GFP gene driven by strong promoter and enhancer sequences allowed an efficient non-lethal transient gene expression assay with an overall transient gene expression frequency of > 4% for uninucleate microspores and between 10 and 20% for binucleate pollen. The technique demonstrated its suitability for analysis of developmental stage-specific gene expression. The assay allowed observation of real-time transgene expression during microspore maturation proving useful for in vitro pollen selection. We have also used this protocol to determine the recombination potential of tobacco male gametic cells by assessing the frequency of extra-chromosomal homologous recombination events after co-delivery of two loss-of-function GFP genes. No increase of extrachromosomal recombination was observed in assays for transient transformation. Key words: Biolistic, GFP, Microspore, Tobacco, Nicotiana tabacum, Transformation D.O.I. 10.3329/ptcb.v19i1.4078 Plant Tissue Cult. & Biotech. 19(1): 9-23, 2009 (June)

scholarly journals Transient GUS gene expression in cassava (Manihot esculenta Crantz) using Agrobacterium tumefaciens leaf infiltration

2014 ◽  
pp. 4338-4349 ◽  
Author(s):  
Paula Díaz T ◽  
Adriana Bernal G ◽  
Camilo López C
Keyword(s):  
Gene Expression ◽  
Agrobacterium Tumefaciens ◽  
Transient Expression ◽  
Manihot Esculenta ◽  
High Efficiency ◽  
Transient Gene Expression ◽  
North Coast ◽  
Manihot Esculenta Crantz ◽  
Cassava Leaves

ABSTRACTObjective. Assess transient gene expression of GUS in cassava (Manihot esculenta Crantz) leaves using Agrobacterium tumefaciens infiltration. Materials and methods. A. tumefaciens strains GV3101 and AGL1 containing pCAMBIA1305.2 were used to evaluate transient gene expression of β-glucuronidase (GUS). A. tumefaciens infiltration (agroinfiltration) was made using both leaves from in vitro and 1 month old greenhouse plants. Leaves were incubated in X-GLUC buffer, stained and photographed to detect GUS activity. Results. Agroinfiltration assays showed GUS transient expression in leaves of cassava varieties widely cultivated in the north coast and eastern savannah, MCOL2215 (Venezuelan) and CM6438-14 (Vergara), respectively. A. tumefaciens agressive strain AGL1 showed high efficiency inducing GUS expression in cassava leaves. Conclusions. We recommend using A. tumefaciens agressive strain AGL1 for agroinfiltration to assess transient expression in cassava leaves.

scholarly journals Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency.

1982 ◽  
Vol 2 (9) ◽  
pp. 1145-1154 ◽  
Author(s):  
Y M Shen ◽  
R R Hirschhorn ◽  
W E Mercer ◽  
E Surmacz ◽  
Y Tsutsui ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Thymidine Kinase ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Transient Gene Expression ◽  
Simian Virus 40 ◽  
Temperature Sensitive ◽  
Enzymatic Function ◽  
Very High

We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (approximately 10(4) base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk-ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.

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