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A new set of recombinant inbred strains complementary to HXB and BXH sets
Experimental Animal Science
A n e w set o f r e c o m b i n a n t i n b r e d strains c o m p l e m e n t a r y to H X B a n d B X H sets VLADIMI'RKILN, VLASTABfLK, RUDOLFKAgPAREK, and DRAHOMfRA Kf~ENOV~I Instituteof Biology and Medical Genetics, 1. MedicalFaculty,Charles University, Prague, Czech Republic
Summary A new set of recombinant inbred [RI] strains has been developed complementary to the original RI strains BXH/HXB using the BXH2 RI strain and the SHR-Lx congenic strain as progenitors, both homozygous in Lx locus. The reason behind the production of this lies in a special SHR/BN gene combination in the BXH2 strain which is increasing the expressivity of the Lx gene and sensitizes this strain to teratogenic effects. On the contrary, the SHR genetic background minimizes the Lx gene expression as well as the teratogenic action of retinoic acid. There are 10 PXO strains in 17~ up to 21 st generation of inbreeding. The variability of leg malformation on PXO strains is similar to that in BXH/HXB polydactylous RI strains. The genotyping of PXO RI set has been started showing relatively high degree of homozygosity [over 90%] in individual RI strains. After inbreeding is accomplished the PXO will have become a useful complement to the original RI strain system. In preliminary teratologic testing, variable sensitivity to retinoic acid has been shown in 3 PXO strains.
Key words: RI strains, Lr locus, genetic background, modifiying genes, teratogen sensitivity Introduction The original BXH and HXB set of recombinant inbred [RI] strains proved to be a useful tool for gene mapping in general (PRAVENECet al. 1996a, b) and for the genetic analysis of cardiovascular and morphologic phenotypes (PRAVENEC et al. 1995, Kf~ENet al. 1996a, b). One of the RI strains -BXH2 and the SHR-Lx congenic strain exhibited strikingly contrasting influences on the manifestation of the polydactyly-luxate syndrome [PLS]. The All procedures have been performed in accordancewith nationalor local animalwelfare legislation. J. Exp. Anim. Sci. 2000; 41:54-56 Urban & FischerVerlag http://www.urbanfischer.de/journals/jeansc 0939-8600/00/41/01-02-054 $12.00/0
A new set of recombinant inbred strains complementary to HXB and BXH sets
55
combination of the SHR and BN modifying genes in the BXH2 strain genetic background led to an extreme PLS morphotype with 4 toes oligodactyly and strong hind zeugopodium affliction together with front feet polydactyly (K0,EN et al. 1996). Moreover, the BXH2 strain was found sensitive to teratogenic effect of Thalidomide (BfLA and Ki~EN 1994) and retinoic acid - RA (BfLA and K ~ N 1996). On the contrary, the SHR genetic background minimizes the Lx gene manifestation to preaxial polydactyly of the hind feet and was found relatively resistant to teratogens. Such a wide difference in modifying influences on malformation mutation and in strain sensitivity to teratogens between BXH2 and SHR-Lx congenic strain inspired us to the development of a new PXO set strains indicated continuous variability of morphological changes following the RA administration. The designation PXO comes from the first capital letters of the phenotypes of hind feet of progenitor strains, which are Polydactylous in the SHR-Lx and Oligodactylous in the BXH2 strain.
Material and M e t h o d s The BXH2 strain and the SHR-Lx congenic strain were mated to produce an F2 population from which randomly chosen pairs were further brother sister mated. At 16th up to 18~ generations of inbreeding, the DNA samples were extracted from the tail skin from 2-3 rats per strain. PCR genotyping is being performed using commercially available primers from Research Genetics according to recommended protocols. For teratological testing the SHR-Lx females were mated overnight with PXO 6, 7 and 10 males and the day on which spermatozoa were detected in vaginal smears was considered as day 1 of pregnancy. The all trans retinoic acid [Sigma] administration, fetuses collection and staining was described earlier (BfLAand KlaN 1996). Fetuses from untreated females served as controls.
Results and Discussion There are 10 PXO RI strains at F17 up to 21 generation of inbreeding. They exhibite variability in PLS manifestation similar to that encountered in HXB and BXH PLS carrying strains. The leg malformation is varrying from 7 toes polydactyly to 4 toes oligodactyly with more or less zeugopodium affliction of the hind feet combined with either normal or afflicted front feet. There still have been fluctuations in the morphotype of individual strains in the number of fingers and zeugopodium affliction. The genotyping using 31 PCR microsatellite markers was performed; albino locus segregation was also recorded. From 320 genotypes evaluated 141 were homozygous in the alleles of SHR origin, 149 were homozygous in the alleles of BN origin and 30 [less than 10%] were heterozygous. The administration of RA to pregnant females revealed the differences in sensitivity of PXO strains as shown on Table 1. In the control group where PXO 6, 7, 10 females were mated with the SHR-Lx male without any treatment we found mostly 6 together with 5 triphalangeal up to 7 toes, no resorptions and mild zeugopodium changes. In the experimental groups after RA administration none fetus has more then 4 toes, what means no overlap with controls. Nevertheless, there is clear cut difference between PXO 6 and PXO 10 hybrid fetuses in the number of the toes. Legs with 1 or 2 toes were prevailing in
56
V. KRENet al.
Table 1. Retinoic acid influence on hind limbs in SHR/PXO, Lx/Lx progeny. RA mg/kg
100.0 100.0 100.0 0.0
PXO x SHR-Lx
6 7 10 6,7,10
Number of hind limbs with
Number of Fetuses Resorptions Litters
7-5T
4
3
2
1 toes
0 0 0 76
0 24 53 0
16 45 50 0
17 30 1 0
31 7 0 0
32 52 52 38
32 44 10 0
7 9 6 4
The females were RA treated on day 13 of pregnancy.
PXO6 while legs with 4 or 3 toes can be seen in PXO10 hybrids. The RA administration has higher embryolethal effect and induced more reduction changes in proximal parts of the hind limbs of PXO6 in comparison with PXO10 hybrid fetuses. The affliction of PXO7 x SHR-Lx fetuses is in between those mentioned above what indicates the continuous variability of the strength of RA teratogenic effect. It can be envisaged that after the PXO RI strains inbreeding is completed and genotyping more progressed, the PXO might help to uncover specific genes or gene combinations involved in gene-teratogen interaction and will have become a useful complement to the original RI strain system.
Acknowledgements Supported by ERBBIO grant 4CT960562, GA CR grants 302/96/0604 and 204/98/K015.
References BfL~, V. and V. KI~,EN.1994. Evidence for teratogenicity of thalidomide using congenic and recombinant inbred rat strains. Fol. Biol.(Praha) 40:161-171. BfLA, V. and V. K~N. 1996. The teratogenic action of retionic acid in rat congenic and recombinant inbred strains. Fol. Biol.(Praha) 42: 167-173. KilN, V. et al. 1996a. Recombinant inbred and congenic strains of the rat for genetic analysis of limb morphogenesis. Fol. Biol.(Praha) 42: 159-168. KfmN, V., D. Ki~ENOV&and V. BfLAet al. 1996b. Recombinant inbred and congenic strains for mapping of genes that are responsible for spontaneous hypertension and other risk factors of cardiovascular disease. Fol. Biol.(Praha)42: 155-158. PRAVENEC, M., D. GAUGUIER,and J. J. SCHOTTet al. 1995. Mapping of quantitative trait loci for blood pressure and cardiac mass in the rat by genome scanning of recombinant inbred strains. J Clin Invest 96: 1973-1978. PRAVENEC, M., V. KNEN, V. BfLA, and D. K~NOVA et al. 1996a. Genetic linkage maps of the rat derived from an intercross and recombinant inbred strains originating from the BN-Lx and SHR progenitors. Fol. Biol.(Praha) 42: 147-154. PRAVENEC, M. et al. 1996b. A genetic linkage map of the rat derived from recombinant inbred strains. Mammal Genome 7:117-127.
Corresponding author: V. KI~,EN,Institute of Biology and Medical Genetics, 1. Medical Faculty, Charles University, Albertov 4, Prague 2, Czech Republic 12800 Tel.: 24914958; Fax: 299492; e-mail: [email protected]
A n e w set o f r e c o m b i n a n t i n b r e d strains c o m p l e m e n t a r y to H X B a n d B X H sets VLADIMI'RKILN, VLASTABfLK, RUDOLFKAgPAREK, and DRAHOMfRA Kf~ENOV~I Instituteof Biology and Medical Genetics, 1. MedicalFaculty,Charles University, Prague, Czech Republic
Summary A new set of recombinant inbred [RI] strains has been developed complementary to the original RI strains BXH/HXB using the BXH2 RI strain and the SHR-Lx congenic strain as progenitors, both homozygous in Lx locus. The reason behind the production of this lies in a special SHR/BN gene combination in the BXH2 strain which is increasing the expressivity of the Lx gene and sensitizes this strain to teratogenic effects. On the contrary, the SHR genetic background minimizes the Lx gene expression as well as the teratogenic action of retinoic acid. There are 10 PXO strains in 17~ up to 21 st generation of inbreeding. The variability of leg malformation on PXO strains is similar to that in BXH/HXB polydactylous RI strains. The genotyping of PXO RI set has been started showing relatively high degree of homozygosity [over 90%] in individual RI strains. After inbreeding is accomplished the PXO will have become a useful complement to the original RI strain system. In preliminary teratologic testing, variable sensitivity to retinoic acid has been shown in 3 PXO strains.
Key words: RI strains, Lr locus, genetic background, modifiying genes, teratogen sensitivity Introduction The original BXH and HXB set of recombinant inbred [RI] strains proved to be a useful tool for gene mapping in general (PRAVENECet al. 1996a, b) and for the genetic analysis of cardiovascular and morphologic phenotypes (PRAVENEC et al. 1995, Kf~ENet al. 1996a, b). One of the RI strains -BXH2 and the SHR-Lx congenic strain exhibited strikingly contrasting influences on the manifestation of the polydactyly-luxate syndrome [PLS]. The All procedures have been performed in accordancewith nationalor local animalwelfare legislation. J. Exp. Anim. Sci. 2000; 41:54-56 Urban & FischerVerlag http://www.urbanfischer.de/journals/jeansc 0939-8600/00/41/01-02-054 $12.00/0
A new set of recombinant inbred strains complementary to HXB and BXH sets
55
combination of the SHR and BN modifying genes in the BXH2 strain genetic background led to an extreme PLS morphotype with 4 toes oligodactyly and strong hind zeugopodium affliction together with front feet polydactyly (K0,EN et al. 1996). Moreover, the BXH2 strain was found sensitive to teratogenic effect of Thalidomide (BfLA and Ki~EN 1994) and retinoic acid - RA (BfLA and K ~ N 1996). On the contrary, the SHR genetic background minimizes the Lx gene manifestation to preaxial polydactyly of the hind feet and was found relatively resistant to teratogens. Such a wide difference in modifying influences on malformation mutation and in strain sensitivity to teratogens between BXH2 and SHR-Lx congenic strain inspired us to the development of a new PXO set strains indicated continuous variability of morphological changes following the RA administration. The designation PXO comes from the first capital letters of the phenotypes of hind feet of progenitor strains, which are Polydactylous in the SHR-Lx and Oligodactylous in the BXH2 strain.
Material and M e t h o d s The BXH2 strain and the SHR-Lx congenic strain were mated to produce an F2 population from which randomly chosen pairs were further brother sister mated. At 16th up to 18~ generations of inbreeding, the DNA samples were extracted from the tail skin from 2-3 rats per strain. PCR genotyping is being performed using commercially available primers from Research Genetics according to recommended protocols. For teratological testing the SHR-Lx females were mated overnight with PXO 6, 7 and 10 males and the day on which spermatozoa were detected in vaginal smears was considered as day 1 of pregnancy. The all trans retinoic acid [Sigma] administration, fetuses collection and staining was described earlier (BfLAand KlaN 1996). Fetuses from untreated females served as controls.
Results and Discussion There are 10 PXO RI strains at F17 up to 21 generation of inbreeding. They exhibite variability in PLS manifestation similar to that encountered in HXB and BXH PLS carrying strains. The leg malformation is varrying from 7 toes polydactyly to 4 toes oligodactyly with more or less zeugopodium affliction of the hind feet combined with either normal or afflicted front feet. There still have been fluctuations in the morphotype of individual strains in the number of fingers and zeugopodium affliction. The genotyping using 31 PCR microsatellite markers was performed; albino locus segregation was also recorded. From 320 genotypes evaluated 141 were homozygous in the alleles of SHR origin, 149 were homozygous in the alleles of BN origin and 30 [less than 10%] were heterozygous. The administration of RA to pregnant females revealed the differences in sensitivity of PXO strains as shown on Table 1. In the control group where PXO 6, 7, 10 females were mated with the SHR-Lx male without any treatment we found mostly 6 together with 5 triphalangeal up to 7 toes, no resorptions and mild zeugopodium changes. In the experimental groups after RA administration none fetus has more then 4 toes, what means no overlap with controls. Nevertheless, there is clear cut difference between PXO 6 and PXO 10 hybrid fetuses in the number of the toes. Legs with 1 or 2 toes were prevailing in
56
V. KRENet al.
Table 1. Retinoic acid influence on hind limbs in SHR/PXO, Lx/Lx progeny. RA mg/kg
100.0 100.0 100.0 0.0
PXO x SHR-Lx
6 7 10 6,7,10
Number of hind limbs with
Number of Fetuses Resorptions Litters
7-5T
4
3
2
1 toes
0 0 0 76
0 24 53 0
16 45 50 0
17 30 1 0
31 7 0 0
32 52 52 38
32 44 10 0
7 9 6 4
The females were RA treated on day 13 of pregnancy.
PXO6 while legs with 4 or 3 toes can be seen in PXO10 hybrids. The RA administration has higher embryolethal effect and induced more reduction changes in proximal parts of the hind limbs of PXO6 in comparison with PXO10 hybrid fetuses. The affliction of PXO7 x SHR-Lx fetuses is in between those mentioned above what indicates the continuous variability of the strength of RA teratogenic effect. It can be envisaged that after the PXO RI strains inbreeding is completed and genotyping more progressed, the PXO might help to uncover specific genes or gene combinations involved in gene-teratogen interaction and will have become a useful complement to the original RI strain system.
Acknowledgements Supported by ERBBIO grant 4CT960562, GA CR grants 302/96/0604 and 204/98/K015.
References BfL~, V. and V. KI~,EN.1994. Evidence for teratogenicity of thalidomide using congenic and recombinant inbred rat strains. Fol. Biol.(Praha) 40:161-171. BfLA, V. and V. K~N. 1996. The teratogenic action of retionic acid in rat congenic and recombinant inbred strains. Fol. Biol.(Praha) 42: 167-173. KilN, V. et al. 1996a. Recombinant inbred and congenic strains of the rat for genetic analysis of limb morphogenesis. Fol. Biol.(Praha) 42: 159-168. KfmN, V., D. Ki~ENOV&and V. BfLAet al. 1996b. Recombinant inbred and congenic strains for mapping of genes that are responsible for spontaneous hypertension and other risk factors of cardiovascular disease. Fol. Biol.(Praha)42: 155-158. PRAVENEC, M., D. GAUGUIER,and J. J. SCHOTTet al. 1995. Mapping of quantitative trait loci for blood pressure and cardiac mass in the rat by genome scanning of recombinant inbred strains. J Clin Invest 96: 1973-1978. PRAVENEC, M., V. KNEN, V. BfLA, and D. K~NOVA et al. 1996a. Genetic linkage maps of the rat derived from an intercross and recombinant inbred strains originating from the BN-Lx and SHR progenitors. Fol. Biol.(Praha) 42: 147-154. PRAVENEC, M. et al. 1996b. A genetic linkage map of the rat derived from recombinant inbred strains. Mammal Genome 7:117-127.
Corresponding author: V. KI~,EN,Institute of Biology and Medical Genetics, 1. Medical Faculty, Charles University, Albertov 4, Prague 2, Czech Republic 12800 Tel.: 24914958; Fax: 299492; e-mail: [email protected]