Research Area:	Cell and Molecular Biology
Technology Exchange:	ES cell / homologous recombination and Transgenesis
Keywords:	Transgenic, Knock-out, Knock-down, Genetically modified, Embryonic Stem cell and Animal Model
Web Links:	Research Group Home Page Service Home Page

Genetically modified models represent one of the most powerful methods of functional gene analysis in vivo. Furthermore, the ability to introduce specific mutations into the genome enables models of human disease to be generated, facilitating insights into the pathophysiology of disease and providing a model with which therapeutic strategies and diagnostic tools can be optimized.

Our group provides groups within Oxford University access to transgenic technologies both on a fee-for-service type arrangement and on a collaborative basis. Technologies offered include embryo microinjection, embryonic stem cell transfection, Knock-out/-in construct design and in vivo shRNA mediated gene Knock-down. In addition, embryo rederivation and cryoconservation services are offered to facilitate the management, transfer and security of genetically modified strains.

The research activity of the group is focused on the development of novel methodologies for the generation of genetically modified models. The aims being to improve the reliability of the technology and to reduce the animal cost of research involving genetically modified models.

There are no collaborations listed for this principal investigator.

Oliver PL, Finelli MJ, Edwards B, Bitoun E, Butts DL, Becker EB, Cheeseman MT, Davies B, Davies KE. 2011. Oxr1 is essential for protection against oxidative stress-induced neurodegeneration. PLoS Genet, 7 (10), pp. e1002338. Read abstract | Read more

Oxidative stress is a common etiological feature of neurological disorders, although the pathways that govern defence against reactive oxygen species (ROS) in neurodegeneration remain unclear. We have identified the role of oxidation resistance 1 (Oxr1) as a vital protein that controls the sensitivity of neuronal cells to oxidative stress; mice lacking Oxr1 display cerebellar neurodegeneration, and neurons are less susceptible to exogenous stress when the gene is over-expressed. A conserved short isoform of Oxr1 is also sufficient to confer this neuroprotective property both in vitro and in vivo. In addition, biochemical assays indicate that Oxr1 itself is susceptible to cysteine-mediated oxidation. Finally we show up-regulation of Oxr1 in both human and pre-symptomatic mouse models of amyotrophic lateral sclerosis, indicating that Oxr1 is potentially a novel neuroprotective factor in neurodegenerative disease. Hide abstract

Chen CM, Krohn J, Bhattacharya S, Davies B. 2011. A comparison of exogenous promoter activity at the ROSA26 locus using a PhiC31 integrase mediated cassette exchange approach in mouse ES cells. PLoS One, 6 (8), pp. e23376. Read abstract | Read more

The activities of nine ubiquitous promoters (ROSA26, CAG, CMV, CMVd1, UbC, EF1α, PGK, chicken β-actin and MC1) have been quantified and compared in mouse embryonic stem cells. To avoid the high variation in transgene expression which results from uncontrolled copy number and chromosomal position effects when using random insertion based transgenic approaches, we have adopted a PhiC31 integrase mediated cassette exchange method for the efficient insertion of transgenes at single copy within a defined and well characterized chromosomal position, ROSA26. This has enabled the direct comparison of constructs from within the same genomic context and allows a systematic and quantitative assessment of the strengths of the promoters in comparison with the endogenous ROSA26 promoter. The behavior of these exogenous promoters, when integrated at ROSA26 in both sense and antisense orientations, reveals a large variation in their levels of activity. In addition, a subset of promoters, EF1α, UbC and CAG, show an increased activity in the sense orientation as a consequence of integration. Transient transfection experiments confirmed these observations to reflect integration dependent effects and also revealed significant differences in the behaviour of these promoters when delivered transiently or stably. As well as providing an important reference which will facilitate the choice of an appropriate promoter to achieve the desired level of expression for a specific research question, this study also demonstrates the suitability of the cassette exchange methodology for the robust and reliable expression of multiple variant transgenes in ES cells. Hide abstract

Davies B, Kirchhoff C. 2011. Adhesion-GPCRs in the Male Reproductive Tract. Adv Exp Med Biol, 706 pp. 179-188. Read abstract | Read more

The male reproductive tract expresses a diverse array of adhesion-GPCRs, many in a highly specific and regulated manner. Despite this specificity of expression, little is known about the function of this receptor family in male reproductive physiology. Insights into function are beginning to emerge with the increasing availability of genetically modified mice harbouring mutations in these genes. Gpr64 is the best characterised of the adhesion-GPCRs in the male reproductive system and the phenotype of Gpr64 knock-out mice implicates this receptor in the regulation of fluid absorption in the efferent ducts and proximal epididymis. This chapter summarizes recent data concerning this receptor and other family members in the male reproductive system. Hide abstract

Dolatshad H, Ahuja A, Hammett M, Preece C, Davies B. 2010. Site specific insertion of genomic transgenes via the PhiC31 integrase TRANSGENIC RES, 19 (2), pp. 327-327.

Waller-Evans H, Prömel S, Langenhan T, Dixon J, Zahn D, Colledge WH, Doran J, Carlton MB, Davies B, Aparicio SA, Grosse J, Russ AP et al. 2010. The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse. PLoS One, 5 (11), pp. e14047. Read abstract | Read more

Adhesion-GPCRs provide essential cell-cell and cell-matrix interactions in development, and have been implicated in inherited human diseases like Usher Syndrome and bilateral frontoparietal polymicrogyria. They are the second largest subfamily of seven-transmembrane spanning proteins in vertebrates, but the function of most of these receptors is still not understood. The orphan Adhesion-GPCR GPR126 has recently been shown to play an essential role in the myelination of peripheral nerves in zebrafish. In parallel, whole-genome association studies have implicated variation at the GPR126 locus as a determinant of body height in the human population. The physiological function of GPR126 in mammals is still unknown. We describe a targeted mutation of GPR126 in the mouse, and show that GPR126 is required for embryonic viability and cardiovascular development. Hide abstract

Baumann C, Davies B, Peters M, Kaufmann-Reiche U, Lessl M, Theuring F. 2007. AKR1B7 (mouse vas deferens protein) is dispensable for mouse development and reproductive success. Reproduction, 134 (1), pp. 97-109. Read abstract | Read more

AKR1B7 (aldo-keto reductase family 1, member 7; also known as mouse vas deferens protein) is a member of the AKR superfamily, and has been suggested to play a role in detoxifying processes on account of its preferred substrates, 4-hydroxynonenal and isocaproaldehyde. High levels of protein expression were found in the vas deferens and the adrenal gland, where sustained expression is dependent on androgen or ACTH respectively. Recently, a remarkable induction of AKR1B7 expression has been reported in the ovary following exogenous injections of LH. In the present study, we confirm this regulation physiologically during the estrous cycle, observing Akr1b7 expression to be restricted to the theca and stromal cells of the proestrus ovary. To further investigate the role of this detoxifying enzyme in both male and female reproduction, we generated knockout mice deficient in AKR1B7. Although AKR1B7 expression in the vas deferens is considerable and tightly regulated in the ovary of wild-type animals, homozygous mutant animals were found to be viable and no reproductive phenotype was observed. Ovarian follicle maturation and spermatozoa parameters remained normal in the absence of this protein. The determination of serum progesterone revealed an increase in hormone concentration in metestrus, while progesterone was found to be decreased in the estrus phase of the cycle in knockout females. Hide abstract

Davies B, Behnen M, Cappallo-Obermann H, Spiess AN, Theuring F, Kirchhoff C. 2007. Novel epididymis-specific mRNAs downregulated by HE6/Gpr64 receptor gene disruption. Mol Reprod Dev, 74 (5), pp. 539-553. Read abstract | Read more

Targeted disruption of the epididymis-specific HE6/Gpr64 receptor gene in mice led to male infertility. In order to characterize the phenotype at a molecular level, we compared the gene expression patterns of wild type (wt) versus knockout (KO) caput epididymides. The caput region of KO males, although morphologically normal, nevertheless showed an aberrant expression pattern. Combining micro array analysis, differential library screening, Northern blot analysis and quantitative RT-PCR, we found that the knockout of the HE6/Gpr64 receptor was mainly associated with the downregulation of genes specific to the initial segment. The list of KO downregulated transcripts comprised Enpp2/autotaxin, the lipocalins 8 and 9, the beta-defensin Defb42, cystatins 8 and 12, as well as the membrane proteins Adam (A Disintegrin And Metalloprotease) 28, claudin-10, EAAC1, and the novel Me9. Clusterin/ApoJ and osteopontin/Spp1 mRNAs, on the other hand, were upregulated in the KO tissues. The Me9 transcript was studied in further detail, and we report here a cluster of related epididymis-specific genes. Me9 is specifically expressed in the initial segment and is representative of a novel and highly conserved mammalian gene family. The family consists of single-exon genes only; intron-containing paralogs have not yet been ascertained. The cloned cDNA sequences predicted hydrophobic polytopic membrane proteins containing the DUF716 motif. Protein expression was shown in the rodent caput epididymidis but remained uncertain in primates. Hide abstract

Kusserow H, Davies B, Hörtnagl H, Voigt I, Stroh T, Bert B, Deng DR, Fink H, Veh RW, Theuring F. 2004. Reduced anxiety-related behaviour in transgenic mice overexpressing serotonin 1A receptors. Brain Res Mol Brain Res, 129 (1-2), pp. 104-116. Read abstract | Read more

Serotonergic neurons play a major role in the modulation of emotion and behaviour. Especially knockout studies have revealed a role for the serotonin(1A) (5-HT(1A)) receptor in anxiety related behaviour. Mutant animals exhibit enhanced anxiety-like responses, possibly resulting from impaired autoinhibitory control of midbrain serotonergic neurons. To further elucidate the role of the 5-HT(1A) receptors in affective behaviour, a complementary approach has been used and transgenic mice overexpressing this receptor subtype have been generated. The expression of the active 5-HT(1A) receptor protein as indicated by autoradiography was transiently increased during early postnatal development (P1.5) as compared to wild-type mice. Within the next 2 weeks, the increase in receptor binding vanished and was also not apparent in adult animals indicating adaptive changes in the regulation of 5-HT(1A) receptor expression. Although no evidence for increased receptor binding in the brains of adult homozygous mice was found by autoradiography, typical phenotypic changes indicative of 5-HT(1A) receptor overactivity were apparent. Transgenic mice revealed a reduced molar ratio of 5-hydroxyindoleacetic acid to serotonin in several brain areas and elevated serotonin values in the hippocampus and striatum. Moreover, anxiety-like behaviour was decreased in male and female transgenic mice and body temperature was lowered in male transgenic mice in comparison with heterozygous and wild-type mice. These findings further underline the pivotal role of 5-HT(1A) receptors in the homeostasis of anxiety-like behaviour and the crucial importance of stimulation of the 5-HT(1A) receptor during the early postnatal development for normal anxiety-like behaviour throughout life. Hide abstract

Davies B, Baumann C, Kirchhoff C, Ivell R, Nubbemeyer R, Habenicht UF, Theuring F, Gottwald U. 2004. Targeted deletion of the epididymal receptor HE6 results in fluid dysregulation and male infertility. Mol Cell Biol, 24 (19), pp. 8642-8648. Read abstract | Read more

Human epididymal protein 6 (HE6; also known as GPR64) is an orphan member of the LNB-7TM (B(2)) subfamily of G-protein-coupled receptors. Family members are characterized by the dual presence of a secretin-like (type II) seven-transmembrane (7TM) domain and a long cell adhesion-like extracellular domain. HE6 is specifically expressed within the efferent ductules and the initial segment of the epididymis, ductal systems involved in spermatozoon maturation. Here, we report that targeted deletion of the 7TM domain of the murine HE6 gene results in male infertility. Mutant mice reveal a dysregulation of fluid reabsorbtion within the efferent ductules, leading to a backup of fluid accumulation in the testis and a subsequent stasis of spermatozoa within the efferent ducts. The fertility phenotype of HE6 knockout mice identifies this receptor as a potential nonsteroidal, nontesticular target for future male contraceptives and identifies an in vivo function for a member of this unusual gene family. Hide abstract