NIEHS Comparative Mouse Genomics Centers Consortium (CMGCC)

Sanchez's Lab


Yolanda Sanchez, Ph.D.: Chk1

CHK1(CHECK1) is an essential checkpoint kinase in mammals and its role in genomic stability is conserved between the yeasts and man. In past months of funding for this project we have embarked on the genetic screen in yeast that yielded hypomorphic alleles of yeast CHK1. For this, we have focused on the non-catalytic domains of Chk1. We have made targeted changes to the yeast codons encoding amino acids that are conserved between the yeast and murine proteins.

Yeast CHK1 alleles

Three deletion mutants and ten point mutants have been generated in the conserved regions of the Chk1 C-terminus. With the exception of chk1-11, 12, 13 and 14 that are to be tested, plasmids carrying mutant chk1 alleles have been transfomed into a chk1?dun1? strain and the resulting transformants have been examined for their sensitivity to hydroxyurea. Two deletion mutants (chk1-2, chk1-8) and possibly three point mutants (chk1-6, chk1-7, chk1-10) display comprised growth on YPD media containing 80 mM HU, while other chk1 mutants show no observed growth defects compared to the wildtype CHK1. The mutant chk1 alleles have also been tested in a cdc13-1 strain, for their ability to initiate and maintain the checkpoint arrest in response to DNA damage (presence of single-strand DNA at telomere proximal regions). A deletion allele, chk1-2, completely abolishes the checkpoint function of Chk1, and strains carrying a second deletion allele chk1-8 , show comprised checkpoint phenotype. Other alleles do not seem to have appreciable defects in checkpoint control. Thus our studies so far suggest that the conserved regions in the Chk1 C-terminus, as defined by chk1-2 and chk1-8, are important in the checkpoint and DNA repair roles of Chk1, and that we will able to make the relevant hypomorphic alleles of the murine homologue that confer reduced activity of MuChk1.

Figure 1: The C-termini of Chk1 homologues and position of changes in the yeast Chk1 protein. Invariant residues among Chk1 homologuess are highlighted in dark boxes. The position of murine Chk1 exons encoding the c-terminal domains is indicated above the amino acid sequence. The underlined region denotes the deleted amino acids in yeast Chk1 that were carried out to generate chk1 deletion alleles. Point mutants are shown with arrows indicating the amino acid substitutions.

Murine Chk1 alleles

Our approach to generate a mouse model will make use of both the Cre/Lox and FLP/FRT systems to study the hypomorphic alleles of Chk1. This combinatorial approach will allow us to study the consequences of carrying the allele as a germline mutation or as a late event by conditional expression of the mutant allele in particular cell types in the adult mouse. Two targeting vectors for making Neo-HPRT/TK-Chk1 ES cell alleles have been constructed. These two constructs have been transfected into ES cells, each resulting approximately 140 positive clones in media containing neomycin and HAT or ganciclovir . For targeting the allele with HPRT marker for negative selection, 120 clones have been screened by the PCR and 1 positive clone has been identified. We are in the process of screening additional clones by the PCR and confirming the clone obtained by the PCR by Southern analyses. The ES clones from the transfection with the construct that has the TK marker for negative selection will be screened in the following weeks.

To rule out a phenotype due to the recombinase sites introduced into the genome as a first step to generate our model, we will generate a mouse carrying the FRT sequences and determine any effect on development or cancer prone phenotype in animals homozygous for this allele. Meanwhile, we are engaged in establishing a strategy to test the hypomorphic alleles identified in yeast and the non synonymous SNP in the kinase domain of Chk1 validated by the Nickerson group in conditional ES cells (next section and Fig. 2 and 3) To introduce the variants into the genome, we will use the ES cells carrying the FRT sites along with the FLPe recombinase (allele with enhanced recombinase activity) to replace the wild-type exons with the mutations that will generate the hypomorphic allele. After careful selection and screening, the ES cells will be injected into blastocysts to generate the mouse model.

Strategy to test Chk1 mutants in ES cells: An Xho I fragment containing the mouse Chk1 cDNA will be cloned into a retroviral expression vector, pMSCVpuro where expression of selectable marker genes and gene of interest are driven by the LTR in order to overcome gene silencing observed after introduction of exogenous genes into ES cells. The non-synonymous SNP will be introduced by PCR mutagenesis. Swapping the PshA I-EcoR I fragment with PCR products containing the desired mutation will introduce mutations in the regulatory domain of Chk1. The resulting pMSCVpuro-Chk1* construct, alone with a packaging vector, pCL-ECO, will be used to transfect the cell line, 293T. The virus, now carrying the mutant Chk1, will be used to transduce ES cells in which one allele of the genomic Chk1 has been disrupted by HPRT and other allele has loxP sites flanking exon 2. Transfection of a Cre plasmid will convert the floxed allele to null allele, thus cells depend on the mutant Chk1 (Chk1*) for growth or viability (Figure 3).

Figure 2: Strategy to generate retroviruses encoding the variants in order to test their function in conditional ES cells.
Figure 3: ES cell specific retroviral system will be used to test the variants in ES cells prior to knocking –in allele.

Future Plans, Key Indicators and up-dated Milestones

The second and next step in our strategy is to rule out a phenotype due to the recombinase sites introduced into the genome as a first step to generate our model. We are generating a targeting construct that contains FRT sites for the FLP recombinase flanking the exons we wish to target in the future. With Dr. Doetschman’s group we will generate a mouse carrying the FRT sequences and determine any effect on development or cancer prone phenotype in animals homozygous for this allele. Once we have determined that the FRT sites do not cause a phenotype we will go back to the ES cells carrying the FRT sites and use the FLPe recombinase (allele with enhanced recombinase activity) to replace the wild-type exons with the mutations that will generate the hypomorphic allele. After careful selection and screening, the ES cells will be injected into blastocysts to generate the mouse model.


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11/29/2002