Mosaic Transgene Expression Research Articles

Comparison of two related lines of tauGFP transgenic mice designed for lineage tracing

BackgroundThe tauGFP reporter fusion protein is produced nearly ubiquitously by the TgTP6.3 transgene in TP6.3 mice and its localisation to microtubules offers some advantages over soluble GFP as a lineage marker. However, TgTP6.3Tg/Tg homozygotes are not viable and TgTP6.3Tg/− hemizygotes are smaller than wild-type. TP6.4 mice carry the TgTP6.4 transgene, which was produced with the same construct used to generate TgTP6.3, so we investigated whether TgTP6.4 had any advantages over TgTP6.3.ResultsAlthough TgTP6.4Tg/Tg homozygotes died before weaning, TgTP6.4Tg/− hemizygotes were viable and fertile and only males were significantly lighter than wild-type. The TgTP6.4 transgene produced the tauGFP fusion protein by the 2-cell stage and it was widely expressed in adults but tauGFP fluorescence was weak or absent in several tissues, including some neural tissues. The TgTP6.4 transgene expression pattern changed over several years of breeding and mosaic transgene expression became increasingly common in all expressing tissues. This mosaicism was used to visualise clonal lineages in the adrenal cortex of TgTP6.4Tg/− hemizygotes and these were qualitatively and quantitatively comparable to lineages reported previously for other mosaic transgenic mice, X-inactivation mosaics and chimaeras. Mosaicism occurred less frequently in TP6.3 than TP6.4 mice and was only observed in the corneal epithelium and adrenal cortex.ConclusionsMosaic expression makes the TgTP6.4 transgene unsuitable for use as a conventional cell lineage marker but such mosaicism provides a useful system for visualising clonal lineages that arise during development or maintenance of adult tissues. Differences in the occurrence of mosaicism between related transgenic lines, such as that described for lines TP6.3 and TP6.4, might provide a useful system for investigating the mechanism of transgene silencing.

Relative transgene expression frequencies in homozygous versus hemizygous transgenic mice

We have used a simple binomial model of stochastic transgene inactivation at the level of the chromosome or transgene, rather than the cellular level, for the analysis of two mouse transgenic lines that show variegated patterns of expression. This predicts the percentages of cells that express one, both or neither alleles of the transgene in homozygotes from the observed percentages of cells, which express the transgene in hemizygotes. It adequately explained the relationship between the numbers of cells expressing the transgene in hemizygous and homozygous mosaic 21OH/LacZ mouse adrenals and mosaic BLG/7 mouse mammary glands. The binomial model also predicted that a small proportion of cells in mosaic mammary glands of BLG/7 homozygotes would express both BLG/7 alleles but published data indicated that all cells expressing the transgene showed monoallelic expression. Although it didn't fit all of the BLG/7 data as precisely as a more complex model, which used several ad hoc assumptions to explain these results, the simple binomial model was able to explain the relationship in observed transgene expression frequencies between hemizygous and homozygous mosaic tissues for both 21OH/LacZ and BLG/7 mice. It may prove to be a useful general model for analysing other transgenic animals showing mosaic transgene expression.

The ROSA26-iPSC Mouse: A Conditional, Inducible, and Exchangeable Resource for Studying Cellular (De)Differentiation

Control of cellular (de)differentiation in a temporal, cell-specific, and exchangeable manner is of paramount importance in the field of reprogramming. Here, we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming, these factors can be replaced by genes of interest-for example, to enhance lineage-directed differentiation-with the use of a trap-coupled RMCE reaction. We show that, similar toESCs, Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon invitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes.

Disrupted-in-Schizophrenia (DISC1) Functions Presynaptically at Glutamatergic Synapses

The pathophysiology of schizophrenia is believed to involve defects in synaptic transmission, and the function of many schizophrenia-associated genes, including DISC1, have been linked to synaptic function at glutamatergic synapses. Here we develop a rodent model via in utero electroporation to assay the presynaptic function of DISC1 at glutamatergic synapses. We used a combination of mosaic transgene expression, RNAi knockdown and optogenetics to restrict both genetic manipulation and synaptic stimulation of glutamatergic neurons presynaptic to other layer 2/3 neocortical pyramidal neurons that were then targeted for whole-cell patch-clamp recording. We show that expression of the DISC1 c-terminal truncation variant that is associated with Schizophrenia alters the frequency of mEPSCs and the kinetics of evoked glutamate release. In addition, we show that expression level of DISC1 is correlated with the probability of glutamate release such that increased DISC1 expression results in paired-pulse depression and RNAi knockdown of DISC1 produces paired-pulse facilitation. Overall, our results support a direct presynaptic function for the schizophrenia-associated gene, DISC1.

56 ESTABLISHMENT OF TRANSGENIC RED FLUORESCENCE PROTEIN (RFP) CLONE DOGS THROUGH A STABLE TRANSMISSION OF RFP GENE TO NEXT GENERATION

Somatic cell nuclear transfer (SCNT) technology has been spotlighted not only for its advantage in producing unlimited numbers of genetically identical animals, but also the possibility of producing complex genetic modifications in animals. However, a few reports showed that mosaic expression of transgene in transgenic animals produced by SCNT (Park et al. 2002) and down-regulated gene expression is sometimes irreversible in their offspring (Bordignon et al. 2003). Therefore, we investigated reproductive ability by a breeding between female transgenic beagles and wild-type beagles. When female transgenic beagles (R1, R2, R3, and R5) expressing red fluorescence protein (RFP) gene reached puberty at 373, 353, 283, and 354 days after birth, serum progesterone concentration was monitored for detecting timing of ovulation. Approximately 72 to 79 h after ovulation, the beagles were naturally mated or artificially inseminated. Pregnancy was confirmed by ultrasonography at Day 30 after insemination. The transgenic bitches (R1, R2, R3, and R5) were then bred with wild-type male dogs, became pregnant, and successfully delivered 13 puppies (9 female and 4 male). In order to prove integration of RFP gene in all offspring, DNA was extracted from the blood of pups on Day 7 after birth. For PCR analysis, a primer pair for the RFP gene, forward primer (5′CGTGAAGCTGAAGGTGA-3′) and reverse primer (5′-CTCGTACTGCTCCACGA-3′), were used to amplify a 517-bp DNA fragment. The initial denaturation was performed at 94°C for 5 min, followed by 30 cycles at 94°C for 40 s (denaturation), 58°C for 40 s (annealing), and 72°C for 40 s (extension), and a final incubation at 72°C for 10 min to ensure complete strand extension. Presence of the RFP transgene in 7 of the puppies was confirmed by PCR and the puppies expressed RFP upon UV illumination. It was not different from the 53.8% expected Mendelian ratio. The present result demonstrated a stable transmission of the RFP gene into 5 female and 2 male offspring in the second generation. Among the second generation, 2 female puppies integrated with the RFP gene were in heat at ∼1-year-old. They were then bred with the semen of a wild-type beagle and bore 6 puppies. In the third generation, 3 puppies carried the RFP gene and results showed the expected Mendelian ratio. In conclusion, the present study demonstrates that female transgenic beagles have normal reproductive ability and a stable insertion of the transgene to the next generation. This study was financially supported by NRF (#M10625030005-508-10N25), SNU foundation (Benefactor; RNL BIO), BK 21 for Veterinary Science, and Purina Korea.

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons. Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages. Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1. Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

Distinct Temporal Requirements for the Homeobox Gene Gsx2 in Specifying Striatal and Olfactory Bulb Neuronal Fates

The homeobox gene Gsx2 (formerly Gsh2) is known to be required for striatal and olfactory bulb neurogenesis; however, its specific role in the specification of these two neuronal subtypes remains unclear. To address this, we have employed a temporally regulated gain-of-function approach in transgenic mice and found that misexpression of Gsx2 at early stages of telencephalic neurogenesis favors the specification of striatal projection neuron identity over that of olfactory bulb interneurons. In contrast, delayed activation of the Gsx2 transgene until later stages exclusively promotes olfactory bulb interneuron identity. In a complementary approach, we have conditionally inactivated Gsx2 in a temporally progressive manner. Unlike germline Gsx2 mutants, which exhibit severe alterations in both striatal and olfactory bulb neurogenesis at birth, the conditional mutants exhibited defects restricted to olfactory bulb interneurons. These results demonstrate that Gsx2 specifies striatal projection neuron and olfactory bulb interneuron identity at distinct time points during telencephalic neurogenesis.

Quantitative proliferation dynamics and random chromosome segregation of hair follicle stem cells

Regulation of stem cell (SC) proliferation is central to tissue homoeostasis, injury repair, and cancer development. Accumulation of replication errors in SCs is limited by either infrequent division and/or by chromosome sorting to retain preferentially the oldest 'immortal' DNA strand. The frequency of SC divisions and the chromosome-sorting phenomenon are difficult to examine accurately with existing methods. To address this question, we developed a strategy to count divisions of hair follicle (HF) SCs over time, and provide the first quantitative proliferation history of a tissue SC during its normal homoeostasis. We uncovered an unexpectedly high cellular turnover in the SC compartment in one round of activation. Our study provides quantitative data in support of the long-standing infrequent SC division model, and shows that HF SCs do not retain the older DNA strands or sort their chromosome. This new ability to count divisions in vivo has relevance for obtaining basic knowledge of tissue kinetics.

Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double‐transgenic pigs

The applicability of tightly regulated transgenesis in domesticated animals is severely hampered by the present lack of knowledge of regulatory mechanisms and the long generation intervals. To capitalize on the tightly controlled expression of mammalian genes made possible by using prokaryotic control elements, we have used a single-step transduction to introduce an autoregulative tetracycline-responsive bicistronic expression cassette (NTA) into transgenic pigs. Transgenic pigs carrying one NTA cassette showed a mosaic transgene expression restricted to single muscle fibers. In contrast, crossbred animals carrying two NTA cassettes with different transgenes, revealed a broad tissue-independent and tightly regulated expression of one cassette, but not of the other one. The expression pattern correlated inversely with the methylation status of the NTA transcription start sites indicating epigenetic silencing of one NTA cassette. This first approach on tetracycline regulated transgene expression in farm animals will be valuable for developing precisely controlled expression systems for transgenes in large animals relevant for biomedical and agricultural biotechnology.

Predictable mosaic transgene expression in ascidian embryos produced with a simple electroporation device

Two customized electroporators were specifically designed for creating transgenic ascidian embryos. These electroporators were simple to build, inexpensive, and produced transgenic embryos with efficiencies that equaled or rivaled commercially available machines. A key design feature of these machines resulted in the generation of consistent electroporation pulses providing repeatability between experiments. These devices were used to optimize experimental parameters allowing for the creation of transient transgenic embryos with predictable patterns of mosaic transgene expression. We used these new electroporators to examine the expression of two different fluorescent protein reporter genes with regard to embryonic cell lineage. In general, transgene expression followed the embryonic cell lineage and coelectroporated transgenes were always expressed in the same embryonic cells. Our analysis also indicated that, during development, transgenes could be lost from embryonic cells, suggesting that transgenes may be present in extrachromosomal arrays, as has been observed in other organisms. Our new electroporator designs will allow ascidian researchers to inexpensively produce transgenic ascidians and should prove useful for adapting the electroporation technique to other marine embryo systems.

Mono-allelic expression of variegating transgene locus in the mouse.

We have generated transgenic mice which express an ovine beta-lactoglobulin transgene during lactation. In two transgenic lines, BLG/7 and BLG/45, beta-lactoglobulin protein levels vary between siblings, reflected at the cellular level by a mosaic transgene expression pattern in the mammary tissue that is reminiscent of position effect variegation. To investigate whether this variegating expression profile can be affected by the introduction of an identical variegating locus on the homologous chromosome, we compared the beta-lactoglobulin expression profiles in mice hemizygous or homozygous for the transgene locus. In BLG/45 mice, milk protein analysis revealed that transgene expression was effectively doubled in homozygous compared to hemizygous mice. In contrast, beta-lactoglobulin protein in hemizygous and homozygous BLG/7 mice displayed a similar range; although minimum expression levels were doubled in the homozygous population, the maximum level of expression was indistinguishable between the two populations. Fluorescent in situ hybridisation (FISH) for transgene mRNA indicated that for a given protein level, the extent of cellular expression is similar in both BLG/7 populations. In homozygous mice genomic DNA and nuclear RNA FISH demonstrated that only one of the two BLG/7 loci is active in expressing cells, while two transcription foci were present in BLG/45 homozygous mice. This mono-allelic transgene expression pattern is not inherited through the germline, as hemizygous mice bred from homozygous parents expressed at the expected hemizygous population level. We discuss these observations in the context of known epigenetic events such as imprinting and trans-inactivation.

Sustained mammary gland-directed, ponasterone A-inducible expression in transgenic mice.

The ability to regulate temporal- and spatial-specific expression of target genes in transgenic mice will facilitate analysis of gene function and enable the generation of murine models of human diseases. The genetic analysis of mammary gland tumorigenesis requires the development of mammary gland-specific transgenics, which are tightly regulated throughout the adult mammary epithelium. Analysis of genes implicated in mammary gland tumorigenesis has been hampered by mosaic transgene expression and the findings that homozygous deletion of several candidate genes (cyclin D1, Stat5A, prolactin receptor) abrogates normal mammary gland development. We describe the development of transgenic mouse lines in which sustained transgene expression was inducibly regulated, both specifically and homogeneously, in the adult mammary gland epithelium. Transgenes encoding RXRalpha and a chimeric ecdysone receptor under control of a modified MMTV-LTR, which targets mammary gland expression, were used. These transgenic 'receptor' lines were crossed with transgenic 'enhancer' lines in which the ecdysone/RXR binding site induced ligand-dependent expression of transgenic beta-galactosidase. Pharmacokinetic analysis of a highly bioactive ligand (ponasterone A), identified through screening ecdysteroids from local plants, demonstrated sustained release and transgene expression in vivo. This transgenic model with both tightly regulated and homogeneous transgene expression, which was sustained in vivo using ligands readily extracted from local flora, has broad practical applicability for genetic analysis of mammary gland disease.

Inducible site-specific somatic mutagenesis in mouse hepatocytes.

To perform ligand-dependent site-specific mutagenesis in the liver, we established transgenic mice expressing the tamoxifen-inducible Cre-ER recombinase (Feil et al., 1996; Brocard et al., 1997) under the control of the human a1-antitrypsin (aAT) promoter (Fig. 1) that is specifically active in hepatocytes (Jallat et al., 1990). Out of 20 transgenic founder animals, three lines expressing Cre-ER in the liver, but not in the other tested organs, were obtained (Fig. 2A and data not shown). The expression pattern of Cre-ER in the liver was analysed in the transgenic line exhibiting the highest Cre-ER expression level. In situ hybridization as well as immunohistochemistry analysis revealed that Cre-ER was expressed in approximately 40%–50% of the hepatocytes (Fig. 2B,C). Mosaic transgene expression using the aAT promoter was previously reported (Ponder, 1996; Veniant et al., 1996). To analyse the recombinase activity, the aATCre-ER transgenic line was crossed with reporter mice containing a loxP-flanked (floxed) allele of a target gene (Feil et al., 1997; Mascrez et al., 1998), and offspring harboring both the Cre-ER and the floxed reporter allele were treated with tamoxifen. Excision occurred for 40%–50% of the reporter allele in the liver and was undetectable in all other organs analysed, as well as in oil-treated animals (Fig. 2D). Thus, recombination occurred in almost all hepatocytes expressing Cre-ER. Taken together, our results show that the aAT-Cre-ER transgenic mice allow conditional site-specific somatic mutagenesis in the liver. The mosaic Cre-ER expression in hepatocytes should be useful to produce genetic chimeras to study gene function during postnatal liver growth, regeneration, and carcinogenesis, and to perform genetic marking studies (Ponder, 1996).

Mosaic expression of an Hprt transgene integrated in a region of Y heterochromatin.

The sensitivity of small transgenes to position effects on their expression suggests that they could serve as indicators of the chromatin properties at their integration site. In particular, they might be expected to provide information on the functional properties of mammalian heterochromatin. We have produced a transgenic line that carries a mouse Hprt minigene on the Y chromosome. In situ hybridization localized the transgene to the heterochromatic portion of the Y. Analysis of transgene expression by isoelectric focusing indicated that the transgene is expressed in a mosaic pattern, and expressing cells have different levels of transgene activity. These findings can be explained as a position effect variegation induced by Y heterochromatin. However, two other transgenes, located at autosomal sites, also showed mosaic activity. If the mosaic transgene expression is attributed to the influence of the chromatin at the insertion site, the Y heterochromatin would appear less potent than some autosomal regions at inducing variegation. An alternative explanation consistent with our results is that the mosaic expression is a semi-autonomous characteristic of these transgene loci. Transgene-expressing and non-expressing cells differed in their ability to grow and be cloned in vitro, indicating that cellular differentiation affected the chromatin structure of the transgene locus on the Y. Karyotype analysis of male mice with the Y-linked transgene and from control male mice carrying the human HPRT transgene, or the mouse Pgk-1 gene at autosomal sites, indicated that the transgene-carrying Y is prone to non-disjunction, generating cells with two (or more) or no Y chromosomes in equal proportion. Further studies will determine if the propensity of this Y chromosome to mitotic errors is also observed in vivo.