Breeding of BER deficient and APOBEC3B expressing mouse strains
We focus on the mechanisms of BER and MMR in the processing of DNA lesions introduced by the AID/APOBEC protein family and how this contributes to development of lymphoid neoplasms due to their role in SHM and CSR.
The Ung and the Smug1 mouse models (Alsøe et al., 2017, Nilsen et al., 2000) are affected in the BER pathway whereas the Msh2 mouse model is affected in the MMR pathway (Kucherlapati et al., 2010). These repair pathways initiate and collaborate in the repair of e.g. uracil in DNA. Uracil may be formed in the genome by deamination either spontaneously or enzymatically by members of the AID/APOBEC family. The APOBEC3B (A3B) mouse model, is a transgenic mouse expressing human A3B (Harris, unpublished), which deaminates cytosine to uracil in the genome and thereby introducing mutations in the genome. We want to examine the effects of BER/MMR processing of these lesions and to pursue molecular and as well as behavioural phenotypes. We will therefore cross the different mouse models to obtain different combinations of the gene modifications.
In order to set up correct breeding and experimental cohorts it is necessary to genotype the mice before they are terminated. For this purpose we will take ear punches of the mice around the time of weening, which at the same time will give an identity to each mouse. During the ear punches the mice will experience mild pain, discomfort and fear but it is not expected to give sustained pain, discomfort or fear for the mice.
The Ung and the Smug1 mouse models do not show signs of disease up to 13 month of age. The same is the case for the A3B mouse model whether on WT or UNG/SMUG1 deficient background, which we have followed up to 12 month of age. Mice older than 12 month will not be used in breeding. The conditional Msh2 mouse model will be bred with an intact MSH2 and are not expected to have any phenotypical problems. The breeding of the Cd19cre mouse model will be less comprehensive but is required to provide mice for the breeding of B-cell specific Msh2 knockout (An experimental FOTS for this cross is in preparation). We do not expect this model to have gross phenotypical problems (despite the B-cell deprivation of the homozygous mouse) as our mice are housed in a MDU unit. In general, animals showing signs of pain, discomfort or disease will be terminated.
The mice are kept as one colony and all genotypes are generated by crossing the mouse strains: Ung-/-, Smug1-/-, A3B-/tg and Msh2LoxP. The strains will regularly be back crossed to avoid genetic drift. In general, the strains will be maintained as heterozygous breeding. However, for breeding of experimental animals, we allow offspring from homozygous breeding as long as the parents are offspring from heterozygous or from heterozygous-homozygous breeding. The colony size applied for is 4300 animals.
The animal experiments cannot be replaced by other means such as cell lines as it will not allow examination of tissue specific, systemic effects on specific tissues or behavioural phenotypes. The colony is managed in a way, which minimizes off targets phenotypes, thereby avoiding the use of animals, which are less likely to show genotype related phenotypes. The mice will be housed at least two mice together and in an enriched environment with access to food and water ad libitum. The mice will not be exposed to interventions except for ear punches. The ear punches are not possible to replace as a tissue biopsy is needed to genotype the mice.
The Ung and the Smug1 mouse models (Alsøe et al., 2017, Nilsen et al., 2000) are affected in the BER pathway whereas the Msh2 mouse model is affected in the MMR pathway (Kucherlapati et al., 2010). These repair pathways initiate and collaborate in the repair of e.g. uracil in DNA. Uracil may be formed in the genome by deamination either spontaneously or enzymatically by members of the AID/APOBEC family. The APOBEC3B (A3B) mouse model, is a transgenic mouse expressing human A3B (Harris, unpublished), which deaminates cytosine to uracil in the genome and thereby introducing mutations in the genome. We want to examine the effects of BER/MMR processing of these lesions and to pursue molecular and as well as behavioural phenotypes. We will therefore cross the different mouse models to obtain different combinations of the gene modifications.
In order to set up correct breeding and experimental cohorts it is necessary to genotype the mice before they are terminated. For this purpose we will take ear punches of the mice around the time of weening, which at the same time will give an identity to each mouse. During the ear punches the mice will experience mild pain, discomfort and fear but it is not expected to give sustained pain, discomfort or fear for the mice.
The Ung and the Smug1 mouse models do not show signs of disease up to 13 month of age. The same is the case for the A3B mouse model whether on WT or UNG/SMUG1 deficient background, which we have followed up to 12 month of age. Mice older than 12 month will not be used in breeding. The conditional Msh2 mouse model will be bred with an intact MSH2 and are not expected to have any phenotypical problems. The breeding of the Cd19cre mouse model will be less comprehensive but is required to provide mice for the breeding of B-cell specific Msh2 knockout (An experimental FOTS for this cross is in preparation). We do not expect this model to have gross phenotypical problems (despite the B-cell deprivation of the homozygous mouse) as our mice are housed in a MDU unit. In general, animals showing signs of pain, discomfort or disease will be terminated.
The mice are kept as one colony and all genotypes are generated by crossing the mouse strains: Ung-/-, Smug1-/-, A3B-/tg and Msh2LoxP. The strains will regularly be back crossed to avoid genetic drift. In general, the strains will be maintained as heterozygous breeding. However, for breeding of experimental animals, we allow offspring from homozygous breeding as long as the parents are offspring from heterozygous or from heterozygous-homozygous breeding. The colony size applied for is 4300 animals.
The animal experiments cannot be replaced by other means such as cell lines as it will not allow examination of tissue specific, systemic effects on specific tissues or behavioural phenotypes. The colony is managed in a way, which minimizes off targets phenotypes, thereby avoiding the use of animals, which are less likely to show genotype related phenotypes. The mice will be housed at least two mice together and in an enriched environment with access to food and water ad libitum. The mice will not be exposed to interventions except for ear punches. The ear punches are not possible to replace as a tissue biopsy is needed to genotype the mice.