Characterization of Transgene IntegrationLociin Transformed Madin Darby Bovine Kidney Cells

Abstract

Generation of transgenic animals is invaluable for both basic and applied research, as it enables the production of biologically active proteins and immunologically compatible organs for xenotransplantation, improvement of livestock production traits, and establishment of animal models of human disease. However, transgene expression is commonly highly variable, even among cell lines independently transformed with the same construct. Consequently, a great number of transfections and screening is needed to achieve transgenic cell lines showing expected phenotype. In this study, we sequenced transgene-host DNA junctions of transgene integration loci in 26 independently transformed Madin Darby bovine kidney (MDBK) cell lines produced by direct liposome transfection. For 15 rescued clones, sequences were of sufficient length and quality to determine unambiguously the position of integration in the bovine genome. Results revealed that transgenes were integrated in 12 different chromosomes, suggesting that there was no chromosomal preference for insertion of exogenous DNA. Most integration events occurred into transcriptionally active regions. No correlation was found between integration into transcribed sequences and the expression level of the beta-gal transgene.

Expression of functional recombinant human factor IX in milk of mice

“…Since this technique results in randomly inserted transgenes, transgenic mice were screened by PCR and confirmed by southern blot analysis in order to evaluate the presence of the transgene introduced in the founders as well as in the progeny. We have studied transgenic integration in bovine cells (Lisauskas et al 2007) to carry out gene-target integration to produce desired recombinant protein expression levels instead of randomly integrated and variable expression.…”

Molecular Characterization of Genetically Manipulated Plants

“…Several approaches, such as plasmid rescue, genomic walking, iPCR and Tail-PCR [5,[15][16][17][18] have been used to determine the plant genome flanking the site of integration of the insert. The general principles of inverse PCR and for Tail-PCR are detailed below.…”

Produção de animais transgênicos por transferência nuclear como modelo de estudo biológico

“…Esta característica permite a quantificação, posicionamento genômico e a confirmação da expressão do transgene (LISAUSKAS et al, 2007), possibilitando o estabelecendo de linhagens de interesse, eventualmente levando ao nascimento de proles exclusivamente portadoras da modificação genética.…”