Doetschman's Lab

Tom Doetschman, Ph.D.

Mouse Modeling Core

1. Tools Mice: Once mouse models are made with human polymorphisms thought to be significant for altered cell cycle and DNA repair function, it is essential that there be in vivo reporter systems to determine the types of mutations that result from the polymorphisms. To this end we have developed two mouse reporter systems. One system involves transgenic mice with the human placental alkaline phosphatase (hPLAP) reporter gene that will revert to full activity after specific types of mutations, namely, frameshifts and transvertions. The second system measures LOH at the Aprt locus. With the first system sections of every tissue in the mouse can be stained for hPLAP reversion mutations, thereby enabling us to compare the frequency and developmental- and tissue-specificity of reversions between wildtype mice and those harboring human polymorphisms in cell cycle regulatory and DNA repair genes. With the second system the effect of these polymorphisms on Aprt LOH can be measured by selection for cells from Aprt+/- mice that have lost function of the wildtype allele. Since the Aprt+/- mice are F1 hybrids of Aprt+/+ and Aprt-/- mice from two inbred strains, strain-specific marker analysis of the Aprt-/- cells enables determination of the type of LOH that has occurred, e.g. chromosomal deletion, large and small deletions within the chromosome, translocations, small mutations within the Aprt gene. These "tools" strains will be submitted to the MMRRC Node Repository Program.
2. Consultation: The Core has met twice with Dr. Knudsen to develop a targeting scheme and construct for introducing polymorphisms in the Rb gene, and it has met several times with Dr. Sanchez for developing a targeting scheme and construct for introducing polymorphisms into the Chk1 gene.
3. Training: The Core has trained Dr. Chen in the Sanchez Lab in ES cell culture and gene targeting techniques. This training was carried out daily for about two months and is now carried out on a "per need" basis.

Interim Progress

The Mouse Modeling Core is designed to make all of the mouse models used by Drs. Stambrook, Sanchez and Knudsen. In addition, the Core works on methodology and reagents to improve the genetic engineering tools. During the past half year the Core has carried out the following tasks.

1. Chk1: Consultation with Drs. Sanchez and Chen for targeting a polymorphism into Chk1. Two constructs with different marker systems have been used for homologous recombination in ES cells. Potentially targeted cells are being screened for each targeting.
2. Chk2: Two consultation sessions between Core personnel and Drs. Stambrook and Penner on targeting strategy have been held.
3. Plk3: Four consultation sessions between Core personnel and Drs. Stambrook and Penner have been held concerning targeting strategies for putting polymorphisms into Plk3. To make the proper targeting construct for Plk3 is complicated by the fact that there is may be another gene within Plk3 and there may also be shared coding sequence. Hence, the targeting construct must be designed so as not to interfere with the other gene.
4. Rb: We have had a consultation session with Dr. Knudsen on targeting a polymorphism into Rb and on the MMTV/CyclinD1b transgenic. When the construct is made we will initiate the gene targeting.
5. Tag & Exchange reagents for engineering polymorphisms (with Stambrook). Since the Cre/LoxP system must introduce LoxP sites into the gene to be modified, there is always the danger that the LoxP sites will interfere with the activity of the target gene. This would result in the inability to distinguish the change in activity caused from the LoxP insertion from that of the polymorphism. To circumvent this problem a Tag & Exchange system is being used which can introduce the polymorphism without any other sequence alterations in the target gene. This system uses a combined positive/negative selection system such that the selectable marker gene replaces the exon of interest (using positive selection) in an initial targeting and is then removed (using negative selection) and replaced by the mutant exon in the second targeting. The bacterial guanine phosphoribosyl transferase (gpt) gene would be ideal for this purpose because it is small (<500 bases) and uses up few restriction sites, as opposed to the mammalian Hprt minigene which is over 3 kb. Since the gpt gene does not express in ES cells, we are re-engineering mammalian coding sequence into the gene so that it may be better expressed. The first attempt at assembling the re-engineered gene was not successful, so alternative approaches are being pursued. When assembled we will begin tested the new gene out in ES cells for positive and negative selection.