Davies Research Group
Research Overview
Transgenic technology enables a gene's role in development, physiology and disease to be investigated and experimentally manipulated in vivo. With this technology, models of human genetic disease can be established by introducing the equivalent human mutation into model organisms. These models can be used to investigate the underlying cause of the disease process and to trial novel therapeutic and diagnostic approaches.
The publication of the human genome sequence and recent genome-wide association studies have identified many genes and mutations whose function has not yet been ascertained, thus it is expected that the use of genetically modified models will increase dramatically over the coming years to address these challenges.
The focus of our group is to increase the efficiency and reliability of the genetic manipulation and to develop novel transgenic methodologies focused at providing in vivo analysis tools for the study of genetic variation and mutation. A further priority for the group is to reduce the animal cost of transgenic research through the generation of more predictable models.
Projects within the group are currently addressing:
- the use of PhiC31 integrase enzymes for the efficient unidirectional insertion of genetic material within specific chromosomal sites.
- the delivery of large chromosomal transgenes as virally packaged DNA
- the use of episomal vectors for transgenic gene delivery
- the use of enzyme assisted DNA cleavage and recombination to allow genetic manipulations to occur in sequences and species which have proven, up to now, resistant to genetic manipulation.
Publications
Chen C-m, 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): e23376. doi:10.1371/journal.pone.0023376
Davies B, Kirchhoff C. Adhesion-GPCRs in the male reproductive tract. 2011. Adv Exp Med Biol. 706:179-88
Waller-Evans H, Prömel S, Langenhan T, Dixon J, Zahn D, Colledge W, Doran J, Carlton M, Davies B, Aparicio S, Grosse J, Russ A. 2010. The Orphan Adhesion-GPCR GPR126 Is Required for Embryonic Development in the Mouse. PLoS ONE 5(11): e14047. doi:10.1371/journal.pone.0014047
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 (Cambridge, England), 134 (1), pp. 97-109. Read abstract | View on PubMed
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. Molecular reproduction and development, 74 (5), pp. 539-53. Read abstract | View on PubMed
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
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. Molecular and cellular biology, 24 (19), pp. 8642-8. Read abstract | View on PubMed
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 research. Molecular brain research, 129 (1-2), pp. 104-16. Read abstract | View on PubMed
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
Ansorge M, Tanneberger C, Davies B, Theuring F, Kusserow H. 2004. Analysis of the murine 5-HT receptor gene promoter in vitro and in vivo. The European journal of neuroscience, 20 (2), pp. 363-74. Read abstract | View on PubMed
The expression level of the 5-HT(1A) receptor gene (htr1a) in the central nervous system (CNS) is implicated in the aetiology and treatment of anxiety disorders and depression. Previous studies of the murine htr1a have revealed that its proximal promoter is GC rich and TATA-less. Several functional transcription factor binding sites, including MAZ and SP1 recognition sequences, have been identified. To further analyse the promoter of this receptor gene, additional upstream sequence information extending to -5.5 kb of the murine htr1a was generated and promoter fragments extending to -20 kb were analysed for activity in cell culture and transgenic animals. Promoter fragments greater than 4.5 kb in length were active in 5-HT(1A) receptor mRNA positive cells and inactive in 5-HT(1A) receptor mRNA negative cells. Smaller fragments were not able to confer this specificity. In agreement, using additive transgenesis to drive LacZ expression in vivo, CNS specific reporter gene expression was found with these longer constructs. Transgene expression in the 4.5- and 20-kb mouse lines resembled the endogenous htr1a expression pattern, whereas the 5.5-kb mouse lines surprisingly revealed strongly reduced expression. None of the three constructs was prone to confer ectopic expression, however, variation of expression between the transgenic lines was observed. Using colocalization studies we analysed the degree of concurrence of transgenic and endogenous htr1a expression brought about by these three different constructs. The highest degrees of colocalization were observed in mice harbouring the 20-kb construct, suggesting a large promoter fragment is required to faithfully direct transgene expression in a 5-HT(1A) receptor like pattern. Hide abstract
Funding Sources
Research within the group is currently funded by the Wellcome Trust and the British Heart Foundation
Research Area
Transgenics
Keywords
Transgenic Knock-out Functional Gene Analysis Knock-down Genetically Modified Model
