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Development of new efficient methods for transgenesis and gene targeting in the rat

Recently, new highly efficient techniques become available for derivation of transgenic or knockout rats for in vivo functional studies of candidate genes for QTL, for analyses of genes with unknown function or for derivation of new rat models of human diseases.

These techniques include transposon mediated transgenesis using microinjections of zygotes with mixes containing Sleeping Beauty constructs and mRNA of hyperactive SB100X transposase. The rate of transgenesis is 14-72% and in most cases only a single insertion outside coding regions is detected. Until recently, gene targeting was impossible in the rat due to the absence of embryonic stem cells. Currently, several new techniques are available for gene targeting that are not based on the use of embryonic stem cells. These techniques include ZFN (Zinc finger nuclease) method, TALEN (Transcription activator-like effector nuclease) method or CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/(CRISPR-associated (Cas)) method. Recently, we derived multiple SHR trangenic lines using the Sleeping Beauty transposon constructs and mRNA of SB100X transposase, for instance lines expressing Venus, Nrf2, Dbh, Pnmt or Endog cDNA under the control of universal and tissue specific promoters. In addition, we were able to obtain the first transgenic rat using the recombinase-mediated cassette exchange (RMCE) method when green fluorescent protein Venus has been exhanged for red fluorescent protein Cherry in a defined position on chromosome 2. We have also derived SHR lines with disrupted Tmem70 gene using ZFN technology or knockouted Plzf gene with the TALEN technique (in collaboration with dr. Zsuzsanna Izsvak (MDC Berlin) and Lajos Mates (Hungarian Academy of Sciences, Szeged)).




Transgenesis with the RMCE method when Venus gene coding for green fluorescent protein was exchanged for the Cherry gene that codes for red fluorescent protein.