Genetic Mapping of Modifiers in Prenatal Lethal Tgfb1 Knockout Mice

Bonyadi, Morteza Jabbarpour (1997) Genetic Mapping of Modifiers in Prenatal Lethal Tgfb1 Knockout Mice. PhD thesis, University of Glasgow.

Abstract

This thesis presents data obtained from genetic analysis of TGFB1 knockout neonates (Shull et al., 1992, Kulkarni et al., 1993) bred onto different genetic backgrounds. TGFB1(-/-) embryos were initially reported to develop to term normally and die by 3 weeks due to multisystemic inflammation (Shull et al., 1992; Kulkarni et al., 1993). Therefore, it was suggested that TGFB1 was not vital during embryogenesis. However, an independent group (Dickson et al., 1995) demonstrated the implication of TGFB1 in an early stage of embryogenesis. It was shown that 50% of the TGFB1(-/-) embryos died in uterus due to defects in yolk sac vasculogenesis and haematopoiesis, whereas the rest did not succumb to the yolk sac phenotype but developed to term normally and died by 3 weeks post-partum as described previously (Kulkarni et al 1993). It has also been shown that maternal TGFB1 can cross the placenta (Letterio et al., 1994). TGFB1 was detected in TGFB1(-/-) embryos born to TGFB1(+/-) females, whereas in those born to null females, TGFB1 was not detected. Thus, it has been suggested that Tgfb1 gene knockout was not equivalent to a protein knockout and the maternally acquired protein could rescue TGFB1(-/-) foetuses and embryos. The work accomplished in this project set out to determine the reason for the existence of at least two different phenotypes; yolk sac insufficiency and survival to birth, among TGFB1(-/-) conceptuses. During this study, the involvement of several possible genetic and/or non-genetic modifying factors in the different expressivity of the TGFB1(-/-) phenotype was examined. The phenotype of TGFB1(-/-) was studied by breeding the Tgfb1 null allele onto two inbred strains; NIH/Ola and C57BI/6J/Ola. Also TGFB1(-/+) heterozygous crosses between various combinations of NIH/Ola and C57BI/6J/Ola enabled study of the possible implications of maternal factors in the different expressivity of the TGFB1(-/-) phenotype. It was estimated that one locus with a codominant pattern of inheritance was responsible for the different expressivity. Due to the codominant behaviour of the modifying gene(s), the F2(NIH/Ola x C57BI/6J/Ola) intercross animals were considered to be the most informative animals for genetic linkage analysis. 50 polymorphic DNA markers were utilised to initiate a genome-wide search by screening 50 TGFB1(-/-) neonates from an F1 intercross. More than 90% of the genome was screened for modifying gene(s) during this study. Four regions of the genome showed suggestive linkage (P<0.05) in the first screen. To confirm the linkages, 30 extra null animals were screened with the interested markers. A small region of mouse chromosome 5 harbouring a genetic modifier met the criterion of definitive linkage (P?10-5). During this project, the feasibility of mapping genetic factors involved in determining early embryonic lethality without need to access the embryos was demonstrated. Fibroblast growth factor receptor type 3 {Fgfr3) and Fibroblast growth factor inducible gene 13 (Fin13), which mapped in the vicinity of the modifying gene, were considered as candidate genes. The expression of these genes were assessed in 9.5 d.pc. embryos and yolk sacs bred onto either NIH/Ola or C57BI/6J/Ola strains by application of RT-PCR. Also expression of Transforming growth factor beta I (Tgfb1) gene was examined in 9.5 d.pc both yolk sac and embryo bred onto either of the strains. By application of heteroduplex analysis (HA) and direct DNA sequencing, the possibility of genetic polymorphism within Fin13 between the two mouse strains was investigated over the coding region. The latter part of the thesis presents data about the genetical mapping of Transforming Growth Factor Beta type II receptor (Tgfbr2) and Plasminogen activator inhibitor type I (PlanHI) on the mouse genome. Following the mapping Tgfbr2 on the mouse genome, two uncloned mouse mutants which mapped in the vicinity of the Tgfbr2 location were examined, by Southern blotting, for the possibility of a large deletion in Tgfbr2 gene in either of these mutants.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Sarah Johnson
• Keywords: Genetics, Developmental biology
• Date of Award: 1997
• Depositing User: Enlighten Team
• Unique ID: glathesis:1997-75941
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 17:14
• Last Modified: 19 Nov 2019 17:14
• URI: https://theses.gla.ac.uk/id/eprint/75941