C53 null is embryonic lethal

High percentage male chimaeric animals (see figure 5.12B) were used to breed to C57/B16 females. Agouti litters, 129 strains are dominant for the agouti allele and C57/B16 are recessive, indicated the mutation passed through the germline. These litters, the N1 generation, were genotyped by southern blot from tail DNA and gave both wild type (+/+) and heterozygous (+/-) animals.

Heterozygous animals from the N1 generation could then be intercrossed to give the N lF l generation, which contained +/+, +/- and homozygous (-/-) animals in the correct mendelian ratio (1:2:1), or backcrossed to C57/B16 animals (N2 generation and so on) to breed the mutation onto a pure bred background. N lF l animals were also genotyped by southern blot from tail DNA. The litters contained only +/+ and +/- animals (First row bold, table 5.13) indicating that in the +/- state, the mutation is viable, but in the -/- state the mutation leads to embryonic lethality. Embryos from various stages were then genotyped to indicate when lethality occurs. To enable the accurate genotyping of embryos, from which very little tissue can be obtained with which to islolate genomic DNA, it was essential to design a robust PCR based genotyping protocol. An effective genotyping strategy can be carried out by using a PCR primer which is specific to the 5’ end of the targeting vector, an endogenous primer 5’ to the targeting vector, and an endogenous primer from the deleted region. The resulting multiplex PCR will give a wild type band, and a mutant band so +/+, +/- and -/- animals can be identified in one PCR (see figure 5.14).

Chapter 5 C53 Targeted mutation

+/+ +/- -/- No. Litters Age

11 25 0 3 N lF l 6 11 2 2 N lF l 7.5dpc 2 13 6 2 N lFl 8.5dpc 4 14 2 2 N lF l 9.5dpc 9 9 5 2 N lF l 10.5dpc 6 17 7 4 N lF l 11.5dpc 6 16 8 3 N lF l 13.5dpc 6 11 4 2 N lF l 14.5dpc 39 91 34 Total 24% 55% 21% % Total

Figure 5.13 No. o f Wild type (+/+) heterozygote (+/-) and homozygote (-/-) animals in N l F l litters.

First line (bold) - 3 litters allowed to proceed to weaning when tail snips were taken for genotyping.

Embryos were dissectedfrom the uterus at various times during gestation and genotyped by PCR from DNA isolated from the embryonic yolk sac. dpc - days post coitum.

Embryos were dissected from the uterus at various stages throughout gestation and genotyped by PCR on genomic DNA isolated from a dissected portion of the embryonic yolk sac. -/- animals were present at all stages investigated (see table 5.13) at the expected Mendelian ratio. Yolk sacs from 8.5dpc -/- embryos and livers from 13.5dpc -/- embryos were taken and RNA was isolated from the tissues to analyse the expression of C53 by RT-PCR. Results confirm there is no expression of C53 in -/- animals as compared to wild type littermates, indicating the targeted mutation is a null (see figure 5.15). The overt phenotype can be seen in figure 5.16. Up to 10.5dpc no overt phenotype can be detected, apart from a slight growth retardation in the -/- embryos. By 12.5dpc the growth retardation is much more

Chapter 5 C53 Targeted mutation

pronounced and embryos are visibly anaemic. At 14.5dpc the anaemia is so

pronounced it is possible to recognise and identify -/- animals before confirmation by genotyping. Wild Type

ft

«

fi

Figure 5.14 €53 PCR genotyping strategy. A Wild type - An endogenous primer 5 ’ to the targeting vector (red), and an endogenous primer from the deleted region (blue) give the wild type band. B Homozygote - PCR primer which is specific to the 5 ’ end o f the targeting vector(yellow) and an endogenous primer 5 ' to the targeting vector (red) give the homozygous band. C The resulting multiplex PCR with all three primers will give a wild type band, and a mutant band so +/+, +/- and - /- animals can be identified in one PCR

Chapter 5 €53 Targeted mutation $ ■t+ _'y< - t+ --f C/Î :/2 À s > >• > > $ 4 a a C53 H PR T

Figure 5.15 RT-PCR analysis o f C53 -/- and +/+ animals. Yolk sac and liver RNA was tested using RT-PCR which indicates there is no C53 expression in null embryos compared to wild type littermates.

Chapter 5 €53 Targeted mutation

10 5dpc

14 5 d p c

Figure 5.16 +/+ and -/- C53 embryos. No phenotype can be seen at 10.5dpc, but by 12.5 dpc anaemia

can be recognised which becomes more pronounced by 14.5dpc 5.2.5 Vascular system and Liver in €53 mutants

Chapter 5 C53 Targeted mutation

To determine if the patterning of the yolk sac the liver was occuring correctly in C53 mutants, the expression of some diagnostic genetic markers expressed in distinct domains within these tissues were examined. FLKl is a receptor tyrosine kinase that is expressed during endothelial cell differentiation (Yamaguchi et al, 1993). It is clear that the expression of this marker is not perturbed in C53 mutant embryos at 8.5dpc suggesting the vasculature is normal (see figure 5.17 A and B). Liver differentiation begins when a region of the cardiac mesoderm signals to the foregut to express hepatocyte specific proteins such as a-fetoprotein (AFP, see Zaret, 1998). By lO.Sdpc, the cells in the foregut diverticulum have expanded into the septum transversum to become a vascularised liver organ, and haematopoietic precursor cells take up residence there. The expression of AFP in C53 mutants is not affected (see figure 5.17 C and D) suggesting the differentiation of the liver hepatocytes occurs as normal. It seems, therefore, that both the vasculature and the embryonic liver develop normally in C53 -/- embryos. This suggests the C53 -/- phenotype is caused by a defect during hematopoiesis and experiments to test the differentiation capacity of haematopoietic system would be needed to confirm this.

Chapter 5 C53 Targeted mutation

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R ip lia

Figure 5.17 Vascular system and Over in C53-/- mutants. FLKl (A and B) and AFP (C and D white

arrowheads) expression appear normal in C53 -/- embryos as compared to wild-type littermates.

Chapter 5 €53 Targeted mutation

5.3 Discussion