Efficient BLG-Cre Mediated Gene Deletion in the Mammary Gland

Using the phage P1-derived Cre/loxP recombination system, we have developed a strategy for efficient mammary tissue specific inactivation of floxed genes. Transgenic mice were generated which express Cre DNA-recombinase under the control of the mammary gland specific promoter of the ovine beta- lactoglobulin (BLG) gene. To test the specificity of Cre mediated recombination, we crossed these mice to animals harbouring a floxed DNA ligase I allele. We show that the BLG-Cre construct specifies mammary specific gene deletion, and furthermore that it is temporally regulated, predominantly occurring during lactation. We fully characterised the extent of gene deletion in one line (line 74). In this strain the virgin gland is characterised by low levels (7%) of Cre mediated deletion, whereas 70–80% of cells within the lactating mammary gland have undergone recombination. Immunohisto-chemistry and indirect in situ PCR were used respectively to demonstrate that both Cre protein and Cre activity were evenly distributed throughout the population of secretory epithelial cells. The level of background recombination in non-mammary tissues was found to be 1.1%, irrespective of mammary gland developmental status. Crossing the transgenic BLG-Cre strain described here to mice harbouring other floxed alleles will facilitate the functional analysis of those genes during differentiation and development of the mammary gland.     

Cre-mediated germline mosaicism: a new transgenic mouse for the selective removal of residual markers from tri-lox conditional alleles

The binary Cre-lox conditional knockout system requires an essential part of the target gene to be flanked by loxP sites, enabling excision in vivo upon Cre expression. LoxP sites are introduced by homologous recombination, together with a selectable marker. However, this marker can disturb gene expression and should be removed. The marker is therefore often prepared with a third, flanking loxP site (tri-lox construct), facilitating its selective removal by partial Cre-lox recombination. We have shown that this excision can be achieved in vivo in the germline using EIIaCre transgenic mice, and have described the advantages of in vivo over in vitro removal. We show here that MeuCre40, a new transgenic mouse, more reliably and reproducibly generates an optimal partial mosaic Cre-lox recombination pattern in the early embryo. This mosaicism was transmitted to the germline and to many other tissues. Alleles with partial deletions, in particular floxed alleles from which the selectable marker was removed, were readily recovered in the next generation, after segregation from the transgene. Segregation via paternal or maternal transmission led to successful recovery of the alleles of interest. We also obtained total deletion of the floxed regions in the same experiment, making this transgene a polyvalent Cre-lox tool. We rigorously tested the ability of MeuCre40 to solve tri-lox problems, by using it for the in vivo removal of neo^R- and hprt-expression cassettes from three different tri-lox mutants.     

Non‐parallel recombination limits cre‐loxP‐based reporters as precise indicators of conditional genetic manipulation

Cre/LoxP‐mediated recombination allows for conditional gene activation or inactivation. When combined with an independent lineage‐tracing reporter allele, this technique traces the lineage of presumptive genetically modified Cre‐expressing cells. Several studies have suggested that floxed alleles have differential sensitivities to Cre‐mediated recombination, which raises concerns regarding utilization of Cre‐reporters to monitor recombination of other floxed loci of interest. Here, we directly investigate the recombination correlation, at cellular resolution, between several floxed alleles induced by Cre‐expressing mouse lines. The recombination correlation between different reporter alleles varied greatly in otherwise genetically identical cell types. The chromosomal location of floxed alleles, distance between LoxP sites, sequences flanking the LoxP sites, and the level of Cre activity per cell all likely contribute to observed variations in recombination correlation. These findings directly demonstrate that, due to non‐parallel recombination events, commonly available Cre reporter mice cannot be reliably utilized, in all cases, to trace cells that have DNA recombination in independent‐target floxed alleles, and that careful validation of recombination correlations are required for proper interpretation of studies designed to trace the lineage of genetically modified populations, especially in mosaic situations. genesis 51:436–442. © 2013 Wiley Periodicals, Inc.     

Efficient in vitro and in vivo excision of floxed sequences with a high-capacity adenoviral vector expressing Cre recombinase

Conditional gene expression or gene disruption using Cre/loxP- or FLP/frt-based recombination systems are valuable tools for studying gene function in development and disease. Recombinant adenoviral vectors expressing Cre recombinase have been suggested as an alternative for deletion of floxed sequences. To further improve this approach we generated a high-capacity adenoviral (HC-Ad) vector expressing Cre (HC-Adcre). In this vector all viral coding sequences are deleted resulting in decreased toxicity. In the present study HC-Adcre efficiently mediated recombination between two loxP sites located in the genome of a reporter cell line. When intravenously injected into ROSA26 reporter mice, a floxed sequence was excised in hepatocytes resulting in expression of the beta-gal reporter. Our data indicate that HC-Ad vectors expressing Cre effectively delete floxed sequences in vivo and have a significant potential as a tool for functional studies in mice.     

Unmodified Cre recombinase crosses the membrane

Site-specific recombination in genetically modified cells can be achieved by the activity of Cre recombinase from bacteriophage P1. Commonly an expression vector encoding Cre is introduced into cells; however, this can lead to undesired side-effects. Therefore, we tested whether cell-permeable Cre fusion proteins can be directly used for lox-specific recombination in a cell line tailored to shift from red to green fluorescence after loxP-specific recombination. Comparison of purified recombinant Cre proteins with and without a heterologous ‘protein transduction domain’ surprisingly showed that the unmodified Cre recombinase already possesses an intrinsic ability to cross the membrane border. Addition of purified recombinant Cre enyzme to primary bone marrow cells isolated from transgenic C/EBPα^fl/fl mice also led to excision of the ‘floxed’ C/EBPα gene, thus demonstrating its potential for in vivo applications. We conclude that Cre enyzme itself or its intrinsic membrane-permeating moiety are attractive tools for direct manipulation of mammalian cells.     

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII−/− transformed fibroblasts showed no influences of TPPII expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.     

Adenovirus mediated homozygous endometrial epithelial Pten deletion results in aggressive endometrial carcinoma

Pten is the most frequently mutated gene in uterine endometriod carcinoma (UEC) and its precursor complex atypical hyperplasia (CAH). Because the mutation frequency is similar in CAH and UEC, Pten mutations are thought to occur relatively early in endometrial tumorigenesis. Previous work from our laboratory using the Pten^+/− mouse model has demonstrated somatic inactivation of the wild type allele of Pten in both CAH and UEC. In the present study, we injected adenoviruses expressing Cre into the uterine lumen of adult Pten floxed mice in an attempt to somatically delete both alleles of Pten specifically in the endometrium. Our results demonstrate that biallelic inactivation of Pten results in an increased incidence of carcinoma as compared to the Pten^+/− mouse model. In addition, the carcinomas were more aggressive with extension beyond the uterus into adjacent tissues and were associated with decreased expression of nuclear ERα as compared to associated CAH. Primary cultures of epithelial and stromal cells were prepared from uteri of Pten floxed mice and Pten was deleted in vitro using Cre expressing adenovirus. Pten deletion was evident in both the epithelial and stromal cells and the treatment of the primary cultures with estrogen had different effects on Akt activation as well as Cyclin D3 expression in the two purified components. This study demonstrates that somatic biallelic inactivation of Pten in endometrial epithelium in vivo results in an increased incidence and aggressiveness of endometrial carcinoma compared to mice carrying a germline deletion of one allele and provides an important in vivo and in vitro model system for understanding the genetic underpinnings of endometrial carcinoma.     

A Novel CCR5 Mutation Common in Sooty Mangabeys Reveals SIVsmm Infection of CCR5-Null Natural Hosts and Efficient Alternative Coreceptor Use In Vivo

In contrast to HIV infection in humans and SIV in macaques, SIV infection of natural hosts including sooty mangabeys (SM) is non-pathogenic despite robust virus replication. We identified a novel SM CCR5 allele containing a two base pair deletion (Δ2) encoding a truncated molecule that is not expressed on the cell surface and does not support SIV entry in vitro. The allele was present at a 26% frequency in a large SM colony, along with 3% for a CCR5Δ24 deletion allele that also abrogates surface expression. Overall, 8% of animals were homozygous for defective CCR5 alleles and 41% were heterozygous. The mutant allele was also present in wild SM in West Africa. CD8+ and CD4+ T cells displayed a gradient of CCR5 expression across genotype groups, which was highly significant for CD8+ cells. Remarkably, the prevalence of natural SIVsmm infection was not significantly different in animals lacking functional CCR5 compared to heterozygous and homozygous wild-type animals. Furthermore, animals lacking functional CCR5 had robust plasma viral loads, which were only modestly lower than wild-type animals. SIVsmm primary isolates infected both homozygous mutant and wild-type PBMC in a CCR5-independent manner in vitro, and Envs from both CCR5-null and wild-type infected animals used CXCR6, GPR15 and GPR1 in addition to CCR5 in transfected cells. These data clearly indicate that SIVsmm relies on CCR5-independent entry pathways in SM that are homozygous for defective CCR5 alleles and, while the extent of alternative coreceptor use in SM with CCR5 wild type alleles is uncertain, strongly suggest that SIVsmm tropism and host cell targeting in vivo is defined by the distribution and use of alternative entry pathways in addition to CCR5. SIVsmm entry through alternative pathways in vivo raises the possibility of novel CCR5-negative target cells that may be more expendable than CCR5+ cells and enable the virus to replicate efficiently without causing disease in the face of extremely restricted CCR5 expression seen in SM and several other natural host species.     

Generation of a germ cell nuclear factor conditional allele in mice

Two recombination steps in embryonic stem (ES) cells were adopted to generate a floxed Germ Cell Nuclear Factor (GCNF) allele. First, a targeting vector containing a loxP site upstream of exon 4, encoding the DNA binding domain (DBD), and a floxed NeoTK double selection cassette downstream of exon 4 was integrated into the GCNF locus by homologous recombination. Second, a Cre-expressing vector was transiently introduced to remove the floxed NeoTK cassette via site-specific recombination. Heterogeneous ES cell populations were found in a single colony after Cre transfection and were separated using an ES cell re-pick step. Floxed GCNF mice were generated and had normal GCNF expression in the adult gonads. Using the Msx2Cre transgenic mice, the floxed GCNF can be completely deleted in the female germline. Taken together, the floxed GCNF mice were successfully generated and female germline deletion of the floxed GCNF allele was achieved using Msx2Cre mice.     

Use of PB-Cre4 Mice for Mosaic Gene Deletion

Transgene expression from short promoters in transgenic animals can lead to unwanted transgene expression patterns, often as a byproduct of random integration of the expression cassette into the host genome. Here I demonstrate that the often used PB-Cre4 line (also referred to as “Probasin-Cre”), although expressing exclusively in the male prostate epithelium when transmitted through male mice, can lead to recombination of loxP-flanked alleles in a large variety of tissues when transmitted through female mice. This aberrant Cre activity due to Cre expression in the oocytes leads to different outcomes for maternally or paternally transmitted loxP-flanked alleles: Maternally inherited loxP-flanked alleles undergo recombination very efficiently, making female PB-Cre4 mice an efficient monoallelic “Cre deleter line”. However, paternally inherited loxP-flanked alleles are inefficiently recombined by maternal PB-Cre4, giving rise to mosaic expression patterns in the offspring. This mosaic recombination is difficult to detect with standard genotyping approaches of many mouse lines and should therefore caution researchers using PB-Cre4 to use additional approaches to exclude the presence of recombined alleles. However, mosaic recombination should also be useful in transgenic “knockout” approaches for mosaic gene deletion experiments.     

Monocarboxylate Transporter 8 Modulates the Viability and Invasive Capacity of Human Placental Cells and Fetoplacental Growth in Mice

Monocarboxylate transporter 8 (MCT8) is a well-established thyroid hormone (TH) transporter. In humans, MCT8 mutations result in changes in circulating TH concentrations and X-linked severe global neurodevelopmental delay. MCT8 is expressed in the human placenta throughout gestation, with increased expression in trophoblast cells from growth-restricted pregnancies. We postulate that MCT8 plays an important role in placental development and transplacental TH transport. We investigated the effect of altering MCT8 expression in human trophoblast in vitro and in a Mct8 knockout mouse model. Silencing of endogenous MCT8 reduced T3 uptake into human extravillous trophoblast-like cells (SGHPL-4; 40%, P<0.05) and primary cytotrophoblast (15%, P<0.05). MCT8 over-expression transiently increased T3 uptake (SGHPL-4∶30%, P<0.05; cytotrophoblast: 15%, P<0.05). Silencing MCT8 did not significantly affect SGHPL-4 invasion, but with MCT8 over-expression T3 treatment promoted invasion compared with no T3 (3.3-fold; P<0.05). Furthermore, MCT8 silencing increased cytotrophoblast viability (∼20%, P<0.05) and MCT8 over-expression reduced cytotrophoblast viability independently of T3 (∼20%, P<0.05). In vivo, Mct8 knockout reduced fetal:placental weight ratios compared with wild-type controls at gestational day 18 (25%, P<0.05) but absolute fetal and placental weights were not significantly different. The volume fraction of the labyrinthine zone of the placenta, which facilitates maternal-fetal exchange, was reduced in Mct8 knockout placentae (10%, P<0.05). However, there was no effect on mouse placental cell proliferation in vivo. We conclude that MCT8 makes a significant contribution to T3 uptake into human trophoblast cells and has a role in modulating human trophoblast cell invasion and viability. In mice, Mct8 knockout has subtle effects upon fetoplacental growth and does not significantly affect placental cell viability probably due to compensatory mechanisms in vivo.     

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene³. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele¹. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton^2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports^2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.     

Hepatic deletion of Smad7 in mouse leads to spontaneous liver dysfunction and aggravates alcoholic liver injury

Background

TGF-β has been known to play an important role in various liver diseases including fibrosis and alcohol-induced fatty liver. Smad7 is an intracellular negative regulator of TGF-β signaling. It is currently unclear whether endogenous Smad7 has an effect on liver function and alcoholic liver damage.

Methodology/Principal Findings

We used Cre/loxP system by crossing Alb-Cre mice with Smad7^loxP/loxP mice to generate liver-specific deletion of Smad7 with loss of the indispensable MH2 domain. Alcoholic liver injury was achieved by feeding mice with a liquid diet containing 5% ethanol for 6 weeks, followed by a single dose of ethanol gavage. Deletion of Smad7 in the liver was associated with increased Smad2/3 phosphorylation in the liver or upon TGF-β treatment in primary hepatocytes. The majority of mice with liver specific deletion of Smad7 (Smad7^liver-KO) were viable and phenotypically normal, accompanied by only slight or no reduction of Smad7 expression in the liver. However, about 30% of Smad7^liver-KO mice with high efficiency of Smad7 deletion had spontaneous liver dysfunction, demonstrated as low body weight, overall deterioration, and increased serum levels of AST and ALT. Degeneration and elevated apoptosis of liver cells were observed with these mice. TGF-β-induced epithelial to mesenchymal transition (EMT) was accelerated in Smad7-deleted primary hepatocytes. In addition, alcohol-induced liver injury and steatosis were profoundly aggravated in Smad7 deficient mice, associated with upregulation of critical genes involved in lipogenesis and inflammation. Furthermore, alcohol-induced ADH1 expression was significantly abrogated by Smad7 deletion in hepatocytes.

Conclusion/Significance

In this study, we provided in vivo evidence revealing that endogenous Smad7 plays an important role in liver function and alcohol-induced liver injury.     

Gene deletion from Plasmodium falciparum using FLP and Cre recombinases: Implications for applied site-specific recombination

The ability to manipulate the genome and induce site-specific recombination using either Flippase (FLP) or Cre recombinase has been useful in many systems including Plasmodium berghei for specific deletion events or to obtain conditional gene expression. To test whether these recombinases are active in Plasmodium falciparum we constructed gene knockouts that contain sequences recognised as templates for site-specific recombination. We tested the ability of FLP and Cre recombinases, expressed conditionally in P. falciparum, to mediate deletion of the human dihydrofolate reductase (hdhfr) drug resistance gene. We show that Cre recombinase is capable of efficient removal of hdhfr by site-specific recombination. In contrast, FLP recombinase is very inefficient, even at the optimum temperature of 30 °C for this enzyme. These results demonstrate that Cre recombinase can be utilised in P. falciparum for deletion of specific sequences such as drug resistance genes. This can be exploited for recycling of drug resistance cassettes and for the design of specific recombination events in P. falciparum.     

Cre activity in fetal albCre mouse hepatocytes: Utility for developmental studies

The albCre transgene, having Cre recombinase driven by the serum albumin (alb) gene promoter, is commonly used to generate adult mice having reliable hepatocyte‐specific recombination of loxP‐flanked (“floxed”) alleles. Based on previous studies, it has been unclear whether albCre transgenes are also reliable in fetal and juvenile mice. Perinatal liver undergoes a dynamic transition from being predominantly hematopoietic to predominantly hepatic. We evaluated Cre activity during this transition in albCre mice using a sensitive two‐color fluorescent reporter system. From fetal through adult stages, in situ patterns of Cre‐dependent recombination of the reporter closely matched expression of endogenous Alb mRNA or protein, indicating most or all hepatocytes, including those in fetal and juvenile livers, had expressed Cre and recombined the reporter. Our results indicate the albCre transgene is effective in converting simple floxed alleles in fetal and neonatal mice and is an appropriate tool for studies on hepatocyte development. genesis 47:789–792, 2009. © 2009 Wiley‐Liss, Inc.     

Population stratification of a common APOBEC gene deletion polymorphism

The APOBEC3 gene family plays a role in innate cellular immunity inhibiting retroviral infection, hepatitis B virus propagation, and the retrotransposition of endogenous elements. We present a detailed sequence and population genetic analysis of a 29.5-kb common human deletion polymorphism that removes the APOBEC3B gene. We developed a PCR-based genotyping assay, characterized 1,277 human diversity samples, and found that the frequency of the deletion allele varies significantly among major continental groups (global F_ST = 0.2843). The deletion is rare in Africans and Europeans (frequency of 0.9% and 6%), more common in East Asians and Amerindians (36.9% and 57.7%), and almost fixed in Oceanic populations (92.9%). Despite a worldwide frequency of 22.5%, analysis of data from the International HapMap Project reveals that no single existing tag single nucleotide polymorphism may serve as a surrogate for the deletion variant, emphasizing that without careful analysis its phenotypic impact may be overlooked in association studies. Application of haplotype-based tests for selection revealed potential pitfalls in the direct application of existing methods to the analysis of genomic structural variation. These data emphasize the importance of directly genotyping structural variation in association studies and of accurately resolving variant breakpoints before proceeding with more detailed population-genetic analysis.     

A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent

Conditional gene inactivation using the Cre/loxP system is widely used, but the difficulty in properly regulating Cre expression remains one of the bottlenecks. One approach to regulate Cre activity utilizes a mutant estrogen hormone-binding domain (ER^T) to keep Cre inactive unless the non-steroidal estrogen analog 4-hydroxytamoxifen (OHT) is present. Here we describe a mouse strain expressing Cre-ER^T from the ubiquitously expressed ROSA26 (R26) locus. We demonstrate efficient temporal and spatial regulation of Cre recombination in vivo and in primary cells derived from these mice. We show the existence of marked differences in recombination frequencies between different substrates within the same cell. This has important consequences when concurrent switching of multiple alleles within the same cell is needed, and highlights one of the difficulties that may be encountered when using reporter mice as indicator strains.     

Split-Cre Complementation Indicates Coincident Activity of Different Genes In Vivo

Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive “split-Cre” fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting.     

Activation of Cre Recombinase Alone Can Induce Complete Tumor Regression

The Cre/loxP system is a powerful tool for generating conditional genomic recombination and is often used to examine the mechanistic role of specific genes in tumorigenesis. However, Cre toxicity due to its non-specific endonuclease activity has been a concern. Here, we report that tamoxifen-mediated Cre activation in vivo induced the regression of primary lymphomas in p53−/− mice. Our findings illustrate that Cre activation alone can induce the regression of established tumors.     

DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages

Sequencing of the 7 kb immC region from four P1-related phages identified a novel DNA recombinase that exhibits many Cre-like characteristics, including recombination in mammalian cells, but which has a distinctly different DNA specificity. DNA sequence comparison to the P1 immC region showed that all phages had related DNA terminase, C1 repressor and DNA recombinase genes. Although these genes from phages P7, ϕw39 and p15B were highly similar to those from P1, those of phage D6 showed significant divergence. Moreover, the D6 sequence showed evidence of DNA deletion and substitution in this region relative to the other phages. Characterization of the D6 site-specific DNA recombinase (Dre) showed that it was a tyrosine recombinase closely related to the P1 Cre recombinase, but that it had a distinct DNA specificity for a 32 bp DNA site (rox). Cre and Dre are heterospecific: Cre did not catalyze recombination at rox sites and Dre did not catalyze recombination at lox sites. Like Cre, Dre catalyzed both integrative and excisive recombination and required no other phage-encoded proteins for recombination. Dre-mediated recombination in mammalian cells showed that, like Cre, no host bacterial proteins are required for efficient Dre-mediated site-specific DNA recombination.