Strong and ubiquitous expression of transgenes targeted into the β-actin locus by Cre/lox cassette replacement

Abstract

Summary: Conventional approaches to produce transgenic mice recurrently yield unpredictable patterns and levels of transgene expression, a situation calling for the development of new techniques to overcome these drawbacks in the context of overexpression studies. Here we present an efficient method for rapid and reproducible transgenesis using the recombinase mediated cassette exchange (RMCE) (Bouhassira et al.: Blood 90:3332-3344, 1997) procedure. A lox511-EGFP-TK/neoloxP cassette was placed under the control of the endogenous mouse b-actin promoter. Heterozygous mice revealed strong and ubiquitous EGFP expression throughout embryogenesis and adulthood. Reproducibly, the same expression pattern was obtained with RMCE when it was used to replace the EGFP-harboring cassette by ECFP or placental alkaline phosphatase (PLAP) reporter genes (DePrimo et al.: Transgenic Res 5:459-466, 1996). Furthermore, the RMCE procedure proved efficient as well in embryonic stem (ES) Key words: AGRP; targeted transgenesis; RMCE; mouse; lox511As a consequence of unpredictable positional effects and varying copy numbers, transgenic mice, generated by conventional DNA injection techniques, commonly present incoherent transgene expression patterns. Therefore, it is often necessary to analyze multiple mouse lines before conclusions, as to the effect of overexpressed transgenic products, can be drawn. In contrast, targeting transgenes into defined and precharacterized loci yields predictable expression patterns due to the invariable transcriptional control exerted by the given endogenous regulatory sequences.In order to reproducibly obtain strong and ubiquitous transgene expression in mice, we have therefore focused investigations on endogenous b-actin, a locus consistently expressed during embryonic development and adulthood. The b-actin genomic region was cloned by screening a BAC library with intron 3 and used to generate the targeting vector by inserting a lox511-EGFPTKNeo-loxP cassette at the b-actin translation start codon in exon 2 (Fig. 1a). Strong EGFP expression was observed in the targeted BALB/c ES-cell clones, as well as in mice which were generated from these ( Fig. 2). Mice heterozygous for the targeted b-actin-EGFP allele were fertile and presented strong, ubiquitous EGFP fluorescence throughout embryogenesis (starting at embryonic day (E)2.5) (Fig. 2b) and in adult organs such as brain, eye, heart, liver, lung, kidney, muscle, thymus, spleen, small intestine, stomach, large intestine, skin, uterus, and tail (Fig. 2a). Except for a 10% lower body weight, X-ray analysis, blood chemo-gram, hemogram, urine analysis, and histopathology all failed to reveal differences with respect to wildtype animals. Homozygous mice, however, were not viable and died between days E10.5 and E11.5 (Fig. 2b) (and as published previously;Shawlot et al., 1998).The b-actin-EGFP ES cells were used to test the efficiency of Cre-recombinase-mediated cassette exchange (RMCE) of lox511-loxP flanked transgenes.Heterospecific lox-sites (wildtype loxP...

Actin isoform expression patterns during mammalian development and in pathology: Insights from mouse models

“…3) [Shawlot et al, 1998]. In a homozygous state, this leads to embryos that die at stage E10.5, which corresponds with the data of Shmerling et al [2005]. In heterozygous mice, b-galactosidase expression was only detected in embryonic stem cells and adult testes, indicating that the phenotype resulting from targeted transgenesis of the bCYA gene is highly dependent on the position of the insert.…”

“…bCYA predominates the newly synthesized mRNAs from the blastocysts, indicating a differential accumulation of bCYA and gCYA mRNAs between maternal and zygotic transcription [Bachvarova et al, 1989]. Information about the expression pattern of bCYA during further development and the adult life came from experiments with transgenic mice, where various reporter genes were expressed under control of the bCYA promoter [Shmerling et al, 2005;Jagle et al, 2007]. These mice show strong and ubiquitous expression of the reporter genes.…”

PITT: Pronuclear Injection-Based Targeted Transgenesis, a Reliable Transgene Expression Method in Mice

“…the phenomenon in which the targeted integration event is not stable is hereafter termed "incomplete exclusiveness of the loxP combination." shmerling et al, created loxP and lox511 docking sites at the β-actin locus following a similar strategy as that of Lauth and coworkers [43], except that they placed the sequences in an inverted orientation to prevent excision of the integrated cassette [62]. They injected the loxP set-containing donor vector together with a recombinant maltose binding protein (MBP)-Cre fusion protein into the pronuclei of zygotes derived from mating of a heterozygous seed male with a wild-type female.…”

“…As described above, two other loci (NGF and β-actin) have been used for Cre-PITT trials [43,62]. Targeted Tg founder mice were successfully obtained at the β-actin locus, although the correct integration efficiency was low (0 to 2.4%).…”

“…The disadvantage in using these loci for PITT is that homozygously tagged mice are not viable, and thus only about half of the fertilized eggs contain a tagged allele, since zygotes are only available from mating between heterozygous males and wild-type females. In addition, the heterozygous KO mice in which the β-actin locus on one allele had been destroyed exhibited abnormal phenotypes [36,62].…”