multiple gene silencing is being required to target and tangle metabolic pathways in eukaryotes and researchers have to develop a subtle method for construction of rna interference (rnai) cassettes. although, several vectors have been developed due to different screening and cloning strategies but still some potential limitations remain to be dissolved. here, we worked out a simple cloning stra...

background human telomerase reverse transcriptase (htert) has become an ideal target for development of anticancer therapy. small interfering rnas (sirnas) are very powerful reagents for gene silencing and show promise for cancer gene therapy. however, only a small number of sirnas have been demonstrated to be effective. for gene therapy targeting htert, it is essential to develop a robust syst...

In a gene targeting experiment, the generation of a targeting construct often requires complex DNA manipulations. We developed a set of cassettes and plasmids useful for creating targeting vectors to modify the mammalian genome. A positive selection marker cassette (PGK/EM7p-npt), which included dual prokaryotic and eukaryotic promoters to permit consecutive selection for recombination in Esche...

Heterologous protein production in yeast expression systems (i.e., Kluyveromyces lactis and Pichia pastoris) normally involves insertion of a linear expression cassette into a target locus in the host genome (1-3). Typically, an expression cassette is assembled in E. coli by first cloning a gene of interest into a circular expression vector (Figure 1A). The expression cassette comprises DNA enc...

In this report, we describe an optimized method for generation of ephA8 BAC transgenic mice expressing the lacZ reporter gene under ephA8 regulatory sequences. First, we constructed a targeting vector that carries a 1.2 kb ephA8 DNA upstream of its first exon, a lacZ expression cassette, a kanamycin cassette, and a 0.7 kb ephA8 DNA downstream of its first exon. Second, the targeting vector was ...

Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RM...

Transcriptional regulation of transgenes depends upon genomic localization in higher eukaryotes. For the applied use of transgenic organisms as producers of pharmaceutically relevant proteins or as pest population control agents, a method to make transgene expression predictable is highly desirable. A targeting method that allows precise cassette replacement comprising solely genes of interest ...

A targeting method to insert genes at a previously characterized genetic locus to make plant transformation and transgene expression predictable is highly desirable for plant biotechnology. We report the successful targeting of transgenes to predefined soybean (Glycine max) genome sites using the yeast FLP-FRT recombination system. First, a target DNA containing a pair of incompatible FRT sites...

The use of bacterial artificial chromosomes (BACs) provides a consistent and high targeting efficiency of homologous recombination in embryonic stem (ES) cells, facilitated by long stretches of sequence homology. Here, we introduce a BAC targeting method which employs restriction fragment length polymorphisms (RFLPs) in targeted polymorphic C57BL/6/Cast/Ei F1 mouse ES cell lines to identify pro...