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PubMed1999

Technical Advance: Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker.

We describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-specific plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/Trps16:PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regenera- tion system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastid-expressed green fluorescent protein was used as a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.

Journal Article↗
PubMed1991

Experimentally introduced defective endogenous proviruses are highly expressed in chickens.

We have previously described the experimental introduction of recombinant subgroup A avian leukosis viruses (ALV) with Rous-associated virus 0 long terminal repeats into the germ line of line 0 chickens and the generation of 23 transgenic lines. Two of these transgenic lines, alv6 and alv11, do not produce infectious virus. Both of these lines contain defective proviruses but do express the gag and/or env protein. We have measured viral RNA expression in tissues derived from alv6, alv11, and the parental line 0. Total RNA was prepared from 9-day embryo, 16-day embryo, 1-day chicken, and 28-day chicken tissues. Viral RNA was detected by Northern RNA transfer analysis. The results indicate that both alv6 and alv11 chickens express viral RNA in all tissues tested regardless of the stage of development. No viral transcripts were detected in any line 0 (C/E; ev-negative) tissue. The levels of biologically active env glycoprotein correlates with the env RNA levels in both lines. In an in vivo interference assay, alv6, alv11, and line 0 chickens were infected with Rous-associated virus 1 and monitored for viremia, antibody against Rous-associated virus 1, and ALV-induced pathogenesis from 4 to 21 weeks. None of the 61 alv6 chickens contained detectable virus or produced antibody against subgroup A ALV. Virus and/or antibody against subgroup A ALV was detected in 34 of the 43 alv11 chickens, whereas 51 of 52 line 0 birds were viremic and/or produced antibody. ALV-induced pathogenesis was observed predominantly in line 0 chickens (10 of 59), whereas very little ALV-induced pathogenesis was seen in either alv6 (1 of 62) or alv11 (1 of 44) chickens. Presumably the mechanism for the increased resistance of alv6 and alv11 chickens was subgroup-specific receptor interference. These results clearly demonstrate that experimentally introduced endogenous proviruses can be expressed at high levels in the avian system.

Animals↗
PubMed2006

Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter.

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.

Animals↗
PubMed2000

The USF proteins regulate transcription of the follicle-stimulating hormone receptor but are insufficient for cell-specific expression.

Expression of the FSH receptor (FSHR) is limited to granulosa cells of the ovary and Sertoli cells of the testis. Previous studies showed that an E box in the proximal promoter of the FSHR gene is required for transcription and that the predominant E box binding proteins are the ubiquitous transcription factors, upstream stimulatory factor 1 (USF1) and USF2. Through cotransfection analysis, we have shown that both wild-type and dominant negative forms of the USF proteins regulate the rat FSHR promoter and that transcriptional activation of FSHR required several domains within the amino-terminal portion of the USF proteins. Analysis of the FSHR promoter region using in vivo genomic footprinting indicated that the E box is occupied by proteins in Sertoli cells but not in cells that fail to express the receptor, despite the presence of the USF proteins. To help delineate the regions of the rat FSHR gene required for correct spatial and temporal expression, transgenic mice harboring two constructs containing variable amounts of 5'-flanking sequence (5,000 bp and 100 bp) were generated. Examination of 16 different transgenic lines revealed varied transgene expression profiles with multiple lines having different amounts of ectopic expression and two lines failing to express the transgene. In addition, little or no expression was observed in Sertoli cells. These studies indicate that additional regulatory sequences outside the region from -5,000 to +123 bp are needed for proper expression in Sertoli cells.

Animals↗
PubMed2003

[Stereoselective determination of propranolol enantiomer in transgenic cell lines expressing human cytochrome P450].

OBJECTIVE: To establish a chiro chromatography for studying the stereoselective metabolism of propranolol (PL) in S(9) incubates prepared from transgenic cell lines expressing human cytochrome P450. METHODS: The concentration of each enantiomer in S(9) incubates was determined through precolumn derivatization with GITC, followed by RP-HPLC assay using S-(+)-propafenone as internal standard. RESULTS: Baseline separations among the diastereomers of S(-)-P, internal standard and R(+)-PL were achieved on Shimpack CLC C(18)ODS column, with UV detection and methanol:water:glacial acetic acid (67/33/0.05,v/v/v) as mobile phase. The assay was simple, accurate, precise and specific. The linear range was from 5 to 500 micromol/L for each enantiomer. The limit of quantitation (LOQ) for the method was 5 micromol/L for the S(-)-and R(+)-PL, respectively (n=5, RSD<10%). The analytical method afforded average recoveries of 98.7 and 98.1% for S(-)- and R(+)-PL, respectively. The reproducibility of the assay was good (RSD<10%). The time-dependent studies showed that PL had the stereoselectivity of S-(-)-isomer in metabolism via CYP2C18 and the stereoselectivity of R-(+)-isomer in metabolism via CYP2C9. CONCLUSION: The method allows to study of stereoselective metabolism of PL in vitro.

Chromatography, High Pressure Liquid↗
PubMed1994

Gamma 2b transgenic mice as a model for the role of immunoglobulins in B cell development.

The development of B lymphocytes is tightly linked to the expression of immunoglobulins (Igs). Pro/preB cells which do not correctly rearrange heavy/light chain genes are aborted. Correctly rearranged Ig transgenes are apparently recognized by the developing B cells and can prevent the rearrangement of endogenous Ig genes. Both mu and gamma 2b heavy chain genes cause this feedback inhibition of heavy chain gene rearrangement. Mu transgenes can in addition replace endogenous MU in its preB cell survival/maturation function. However, several different transgenic lines have shown that gamma 2b transgenes do not provide the nurturing functions of mu, except for one unique gamma 2b transgenic line, the C line. In this line mature B cells express gamma 2b only. Presumably, an unknown gene has been activated at the transgene integration site whose product overcomes the need for mu. The function of this gene depends of the presence of the surrogate light chain (sL), and thus must operate in combination with the preB cell receptor or in a downstream signaling/antiapoptosis event requiring the gamma 2b/sL receptor. The analysis of the two types of gamma 2b transgenic mice shows that the signals for preB cell development are highly complex and promises to reveal new insights into the molecular and cellular mechanisms of B cell maturation.

Animals↗
PubMed2001

A probasin-large T antigen transgenic mouse line develops prostate adenocarcinoma and neuroendocrine carcinoma with metastatic potential.

Neuroendocrine (NE) cells may be involved not only in growth and differentiation of the normal prostate but also in carcinogenesis and progression of prostate adenocarcinoma (Pca), including development of androgen resistance. However, the exact pathophysiology of NE cells in Pca remains poorly understood. Here we describe a transgenic model of Pca with progressive NE differentiation. Seven lines of transgenic mice with the rat prostate-specific large probasin promoter linked to the SV40-large T antigen (Tag) that develop prostatic neoplasia have been established. In this study, one of the seven lines (12T-10) was characterized by examination of 52 mice aged from 2-12 months. With advancing age, low-grade prostatic intraepithelial neoplasia, high-grade prostatic intraepithelial neoplasia, microinvasion, invasive carcinoma, and poorly or undifferentiated carcinoma with NE differentiation appeared in the prostates in sequential order. Whereas Tag is expressed uniformly in prostate epithelium, only an increasing subset of cells in prostatic intraepithelial neoplasia showed NE differentiation by chromogranin immunostaining. Frankly invasive carcinoma developing subsequently showed occasional definitive glandular differentiation (adenocarcinoma) and particularly undifferentiated carcinoma with NE histological features similar to those observed in NE carcinomas in humans. The NE carcinomas occurred in the dorsolateral and ventral lobes and were generally androgen receptor negative. Twenty-one of 32 (66%) mice aged > or = 6 months and 15 of 17 (88%) mice aged > or = 9 months developed metastatic tumors, as confirmed by histology and/or Tag immunohistochemistry. Metastases occurred at the later time points, with metastasis to regional lymph nodes, liver, and lung being particularly common. Metastases showed histological features of NE differentiation, as confirmed by chromogranin immunostaining and electron microscopy. An athymic nude mouse that received a s.c. implant of a primary NE tumor developed Tag-positive metastatic tumors with similar NE differentiation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified identical protein profiles between the primary NE tumor and lesions in the extraprostatic organs. Hence, in the 12T-10 large probasin promoter-Tag mouse, high-grade prostatic intraepithelial neoplasia develops progressively greater NE differentiation and progresses to invasive adenocarcinoma and NE carcinoma, with a high percentage of metastases. The predictable progression through these stages will allow testing of therapeutic interventions as well as possible further delineation of the role of NE cells in Pca progression.

Adenocarcinoma↗
PubMed2003

Recapitulation of fast skeletal muscle development in zebrafish by transgenic expression of GFP under the mylz2 promoter.

A 1,934-bp muscle-specific promoter from the zebrafish mylz2 gene was isolated and characterized by transgenic analysis. By using a series of 5' promoter deletions linked to the green fluorescent protein (gfp) reporter gene, transient transgenic analysis indicated that the strength of promoter activity appeared to correlate to the number of muscle cis-elements in the promoter and that a minimal -77-bp region was sufficient for a relatively strong promoter activity in muscle cells. Stable transgenic lines were obtained from several mylz2-gfp constructs. GFP expression in the 1,934-bp promoter transgenic lines mimicked well the expression pattern of endogenous mylz2 mRNA in both somitic muscle and nonsomitic muscles, including fin, eye, jaw, and gill muscles. An identical pattern of GFP expression, although at a much lower level, was observed from a transgenic line with a shorter 871-bp promoter. Our observation indicates that there is no distinct cis-element for activation of mylz2 in different skeletal muscles. Furthermore, RNA encoding a dominant negative form of cAMP-dependent protein kinase A was injected into mylz2-gfp transgenic embryos and GFP expression was significantly reduced due to an expanded slow muscle development at the expense of GFP-expressing fast muscle. The mylz2-gfp transgene was also transferred into two zebrafish mutants, spadetail and chordino, and several novel phenotypes in muscle development in these mutants were discovered.

Animals↗
PubMed1998

Cre expression in primary spermatocytes: a tool for genetic engineering of the germ line.

Transgenic mice were generated expressing a testicular Cre recombinase driven by promoter sequences derived from the gene encoding Synaptonemal Complex Protein 1 (Sycp1), expressed at an early stage of the male meiosis (leptotene to zygotene). Recombination at target LoxP sites was examined during germinal differentiation in mice harboring Sycp1-Cre and a second transgene where LoxP sites flank either the beta geo coding region, the Pgk1 promoter, or a tk-neo cassette inserted into the Rxr alpha locus. The LoxP-flanked transgenes were stably maintained in the somatic tissues of the double transgenic animals, as well as in the progeny of the females. Mice born after mating the double-transgenic males with normal females showed extensive deletions of the LoxP-flanked sequences. When the males were hemizygous for the Sycp1-Cre transgene, the deletions were observed even in the fraction of the offspring which had not inherited the Cre gene, thus demonstrating that expression occurred in the male parent during spermatogenesis. The high efficiency of excision at the LoxP sites makes the Sycp1-Cre transgenic males suitable for evaluating the role of defined gene functions in the germinal differentiation process.

Animals↗
PubMed2003

Transgenic planarian lines obtained by electroporation using transposon-derived vectors and an eye-specific GFP marker.

To generate transgenic planarians we used a set of versatile vectors for animal transgenesis based on the promiscuous transposons, mariner, Hermes and piggyBac, and a universal enhanced GFP (EGFP) marker system with three Pax6 dimeric binding sites, the 3xP3-EGFP developed by Berghammer et al. [Berghammer, A. J., Klinger, M. & Wimmer, E. A. (1999) Nature 402, 370-371]. This marker is expressed specifically in the eyes of various arthropod taxa. Upon microinjection into the parenchyma of adult planarians and subsequent electroporation, these vectors transpose efficiently into the planarian genome. One of the cell types transformed are the totipotent "neoblast" stem cells present in the adults, representing 30% of total cells. The neoblast represents a unique cell type with the capacity to proliferate and to differentiate into all somatic cell types as well as into germ cells. All three transposon vectors have high transformation efficiency, but only Hermes and piggyBac show stable integration. The mariner vector is frequently lost presumably because of the presence of active mariner-type transposons in the genome of the Girardia tigrina. Transformed animals are mosaics containing both transformed and untransformed neoblasts. These differentiate to form EGFP-positive and -negative photoreceptor cells. Such mosaicism is maintained through several cycles of regeneration induced by decapitation or asexual reproduction. Transformed neoblasts also contribute to the germ line, and can give rise to pure transgenic planarian lines in which EGFP is expressed in all photoreceptor cells after sexual reproduction. The presence of the transgenes was confirmed by PCR, plasmid rescue assay, inverse PCR, and Southern blotting. Our results with the 3xP3-EGFP marker confirm the presence of Pax6 activity in the differentiated photoreceptor cells of planarian eyes. Transgenesis will be an important tool to dissect developmental molecular mechanisms in planarian regeneration, development and stem cell biology, and may also be an entry point to analyze the biology of parasitic Platyhelminthes.

Animals↗
PubMed2006

Metabolic changes in Arabidopsis thaliana expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D.

Anthranilate synthase (AS) is a key enzyme in tryptophan (Trp) biosynthesis. Metabolic changes in transgenic Arabidopsis plants expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D were investigated with respect to Trp synthesis and effects on secondary metabolism. The Trp content varied depending on the transgenic line, with some lines showing an approximately 200-fold increase. The levels of AS activity in crude extracts from the transgenic lines were comparable to those in the wild type. On the other hand, the enzyme prepared from the lines accumulating high levels of Trp showed a relaxed feedback sensitivity. The AS activity, determined in the presence of 50 microM L-Trp, correlated well with the amount of free Trp in the transgenic lines, indicating the important role of feedback inhibition in control of Trp pool size. In Arabidopsis, Trp is a precursor of multiple secondary metabolites, including indole glucosinolates and camalexin. The amount of indol-3-ylmethyl glucosinolate (I3 M) in rosette leaves of the high-Trp accumulating lines was 1.5- to 2.1-fold greater than that in wild type. The treatment of the leaves with jasmonic acid resulted in a more pronounced accumulation of I3 M in the high-Trp accumulating lines than in wild type. The induction of camalexin formation after the inoculation of Alternaria brassicicola was not affected by the accumulation of a large amount of Trp. The accumulation of constitutive phenylpropanoids and flavonoids was suppressed in high-Trp accumulating lines, while the amounts of Phe and Tyr increased, thereby indicating an interaction between the Trp branch and the Phe and Tyr branch in the shikimate pathway.

Anthranilate Synthase↗
PubMed2001

Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.

The critical shortage of human donor organs has generated growing interest for porcine to human xenotransplantation. The major immunological barrier to xenotransplantation is the hyperacute rejection (HAR) response that is mediated by preformed xenoreactive antibodies and complement. A promising strategy to control the complement activation, is the expression of human complement regulatory proteins in transgenic animals. We have used the human early cytomegalovirus (CMV) promoter to drive expression of the human complement regulatory protein CD59 (hCD59) in transgenic pigs. A total of eight live transgenic founder animals was born from which five transgenic lines could be established. mRNA analysis and Western blotting revealed high expression of hCD59 in heart, kidney, skeletal muscle, and skin in animals of lines 1 and 5, as well as in the pancreas of four lines. This pattern of expression was confirmed by immunhistological staining. A cell-specific expression in heart and kidney tissue of transgenic lines 1 and 5 was determined. Primary fibroblasts and endothelial cell cultures derived from the aorta of transgenic pigs showed a significantly diminished sensitivity against the challenge with xenoreactive human antibodies and complement whereas non-transgenic control cells were highly susceptible to complement mediated lysis. Ex vivo perfusion of kidneys with pooled human blood revealed a significant protective effect of hCD59 against HAR. The average survival of transgenic kidneys was significantly extended (P<0.05) over nontransgenic controls (207.5+/-54.6 vs. 57.5+/-64.5 min). These data support the concept that hCD59 protects nonprimate cells against human complement mediated lysis and suggest that donor pigs transgenic for hCD59 could play a crucial role in clinical xenotransplantation. Two of five hCD59 transgenic lines showed strong hCD59 expression in several organs relevant for xenotransplantation and a protective effect against HAR. This indicates that the use of the CMV-promoter can facilitate the selection process for optimized transgene expression.

3T3 Cells↗
PubMed1998

Transgenes are dispensable for the RNA degradation step of cosuppression.

Cosuppression results in the degradation of RNA from host genes and homologous transgenes after transcription in the nucleus. By using grafting experiments, we have shown previously that a systemic signal mediates the propagation of cosuppression of Nia host genes and 35S-Nia2 transgenes from silenced 35S-Nia2 transgenic stocks to nonsilenced 35S-Nia2 transgenic scions but not to wild-type scions. Here, we examined the requirements for triggering and maintenance of cosuppression in various types of scions. Grafting-induced silencing occurred in 35S-Nia2 transgenic lines over-accumulating Nia mRNA whether they are able to spontaneously trigger cosuppression or not and in 35S-Nia2 transgene-free plants over-accumulating host Nia mRNA caused by metabolic derepression. When grafting-induced silenced scions were removed from the silenced stocks and regrafted onto wild-type plants, silencing was not maintained in the 35S-Nia2 transgene-free plants and in the 35S-Nia2 transgenic lines that are not able to trigger cosuppression spontaneously. Conversely, silencing was maintained in the 35S-Nia2 transgenic lines that are able to trigger cosuppression spontaneously. Our results indicate that the presence of a 35S-Nia2 transgene is dispensable for the RNA degradation step of posttranscriptional silencing when host Nia mRNA over-accumulate above the level of wild-type plants. They also suggest that grafting-induced RNA degradation does not result in the production of the systemic silencing signal required for spontaneous triggering and maintenance.

Genes, Plant↗
PubMed2002

Generation and characterization of two transgenic mouse lines expressing human ApoE2 in neurons and glial cells.

Apolipoprotein E (apoE) isoforms are key determinants of susceptibility to late-onset Alzheimer's disease (AD). The epsilon 4 and epsilon 2 alleles have been associated with increased and decreased risk for AD, respectively. We have generated and characterized transgenic mice in which the human apoE2 gene is expressed under the control of the platelet-derived growth factor B-chain (PDGF-B) promoter, or the transferrin (TF) promoter. S1 nuclease analysis and immunoblotting showed that the PDGF-B apoE2 mice express apoE2 exclusively in the brain whereas the TF apoE2 mice express apoE2 in the liver and in the brain. In the TF apoE2 mouse line, apoE2 is also detected in the plasma. The PDGF-B apoE2 and the TF apoE2 transgenic mice were bred back to apoE(-)(/)(-) background. Immunohistochemical analysis showed that the PDGF apoE2 x apoE(-)(/)(-) and the TF apoE2 x apoE(-)(/)(-) mice express human apoE2 within the neocortex in hippocampal neurons and glial cells, respectively. ApoE(-)(/)(-) mice have been shown to develop age-dependent loss of synaptophysin. Immunoblotting of mouse brain extracts and immunohistochemical analysis of brain sections showed that apoE expression in both apoE2 x apoE(-)(/)(-) transgenic lines was associated with significant recovery of brain synaptophysin levels as compared to the levels of apoE(-)(/)(-) littermates of the same age. These apoE2-expressing mice, when bred back on amyloid precursor protein (APP) transgenic mice or other mouse lines featuring alterations in lipoprotein metabolism, may provide new mouse models for elucidating the role of apoE2 in lipid homeostasis in the brain and in the pathogenesis of AD.

Animals↗
PubMed1991

A transgenic mouse line developed to express human amyloidogenic transthyretin cDNA in the brain.

To elucidate the role of amyloid deposits in the pathogenesis of degenerative disorders of brain, we attempted to develop transgenic mice producing amyloidogenic human variant transthyretin (TTR) in the brain. A minigene, in which the promoter region of the mouse myelin basic protein (MBP) gene was ligated to the human mutant TTR cDNA, was used for microinjection. A transgenic mouse line expressing the human TTR mRNA specifically in the brain was thus derived. However, human TTR was not detected in the brain or in the serum of these mice. These data suggest that the human mutant TTR mRNA is not efficiently translated in the mouse brain.

Amino Acid Sequence↗
PubMed2000

Evaluation of potato tuber moth (Lepidoptera: Gelechiidae) resistance in tubers of Bt-cry5 transgenic potato lines.

The potato tuber moth, Phthorimaea operculella (Zeller), in tropical and subtropical countries, is the most destructive pest of potato, Solanum tuberosum L. The larvae attack foliage and tubers in the field and in storage. The purpose of this study was to evaluate the efficacy of a Bt-cry5 transgene to control the potato tuber moth in tuber tissues. Tuber bioassays using stored (11-12 mo old) and newly harvested tubers of Bt-cry5-Lemhi Russet and Bt-cry5-Atlantic potato lines showed up to 100% mortality of 1st instars. Mortality was lowest in the newly harvested tubers of Bt-cry5-Atlantic lines (47.1-67.6%). Potato tuber moth mortality was 100% in the Bt-cry5-Spunta lines that were transformed with Bt-cry5 gene controlled by the CaMV 35S promoter (pBIML5 vector) and in 2 of 3 lines transformed with Bt-cry5 gene controlled by the Gelvin super promoter (pBIML1 vector). The transgenic Spunta lines expressing Bt-cry5 controlled by the patatin promoter (pBMIL2 vector) showed the lowest tuber moth mortality (25.6 and 31.1%). The Bt-cry5 transgenic lines with high tuber expression of B. thuringiensis have value in an integrated pest management system to control potato tuber moth.

Animals↗
PubMed2006

Nestin-Cre transgenic mouse line Nes-Cre1 mediates highly efficient Cre/loxP mediated recombination in the nervous system, kidney, and somite-derived tissues.

Here we describe the generation of the Nes-Cre1 transgenic mouse line in which Cre recombinase expression is controlled by the rat nestin promoter and intron 2 enhancer. This line has previously been used for conditional loss-of-function studies of various genes in the central nervous system and first branchial arch ectoderm. Here we report the detailed temporal and spatial recombination pattern of Nes-Cre1 using three different reporters of Cre-mediated recombination, ROSA26R (R26R), Z/AP, and Z/EG. Cre/loxP recombination was detected in embryos as early as the head-fold stage. By embryonic day (E)15.5 recombination occurred in virtually all cells of the nervous system and unexpectedly also in somite-derived tissues and kidneys. Tissues with little or no recombination included heart, liver, thymus, and lung. This study suggests that Nes-Cre1-mediated recombination occurs in progenitor cell types present in the neuroectoderm, the developing mesonephros, and the somites.

Animals↗
PubMed2003

The insulator effect of the 5'HS4 region from the beta-globin chicken locus on the rabbit WAP gene promoter activity in transgenic mice.

Previous studies have shown that the 5'HS4 DNaseI hypersensitive site of the chicken beta-globin locus is endowed with classic insulator activities: (i) it blocks the interaction between promoter and enhancers when it is inserted between them (ii) it confers expression of integrated foreign genes independent of their position in the chromatin. The aim of this present work was to determine whether the 5'HS4 element was able to stimulate the expression level and/or to increase the expression frequency of a luc+ reporter gene controlled by the rabbit WAP gene promoter. Two constructs with 5'HS4 insulator (p5'HS4-WAPluc) or without (pWAPluc) were introduced in mouse fertilised oocytes. All transgenic lines containing the 5'HS4 element (six lines) expressed the transgene whereas only two out of eight lines harbouring the pWAP-luc construct expressed the transgene to a significant level. Moreover, the mean level of expression was seven times higher in p5'HS4WAP-luc lines than in pWAP-luc lines. Even all these benefits on transgene expression, the 5'HS4 element did not confer a copy-dependent expression, did not decrease the ectopic expression of the reporter gene and did not decrease the variability of expression. Thus, the 5'HS4 element does not have all the properties of a perfect insulator on a construct containing the luc+ reporter gene controlled by the rabbit WAP promoter.

Animals↗