Center for Integrated Animal Genomics Research Experience in Molecular Biotechnology & Genomics Summer 2007 Kaizan J. Kalyaniwalla 1, Rafi Awedikian 2, and N. Matthew Ellinwood 2. 1 Biochemistry, University of Wisconsin-Madison, 2 Department of Animal Science, Iowa State University Program supported by the National Science Foundation Research Experience for Undergraduates DBI Production of a Conditional Knockout Mouse Model for the Deficient Enzyme in Mucopolyaccharidosis IIIC: Heparin Acetyl-coenzyme A:α-glucosaminide N-acetyltransferase Introduction The mucopolysaccharidoses (MPS): a group of lysosomal storage enzymopathies defined by glycosaminoglycan (GAG) accumumulation. Accumulation of GAGs within the lysosome: leads to progressive cellular, organ, and system damage, affecting primarily bones and/or the central nervous system. The MPSs: 7 clinical syndromes comprised of 11 enzymopathies. Severe forms are characterized by pre-mature death, often in the pediatric period. MPS III: four known subtypes, each caused by deficiency of one of four enzymes critical to heparan sulfate degradation (fig.2). MPS IIIC: autosomal recessive deficiency of a lysosomal membrane enzyme (heparin acetyl-coenzyme A:α-glucosaminide N-acetyltransferase (HGSNAT, N-acetyltransferase)). To date no animal models of MPS IIIC exist for clinical and pathology investigations.  N-acetyltransferase:  Last MPS causing gene to be mapped and characterized.  Unique: only non-hydrolytic, membrane-bound MPS associated enzyme.  Prototype of new class of enzyme that transport activated acetyl residues across cell membranes (Hrebicek et.al 2006).  MPS IIIC patients: carry out acetyl-CoA/CoA exchange, but no transfer of bound acetyl group to glucosamine (Ausseil et.al 2005). Figure 3. a) Region of HGSNAT gene from exons 4-6, with fragments A, B, and C indicated. b) Methodology for cloning fragments A-C into pBY49a vector. c) HGSNAT gene in murine embryonic stem cells (exons 4-6). d) Homologous recombination between HGSNAT and targeting vector containing TK negative selection marker, neomycin positive selection marker, and FRT and LoxP sites. e) FRT sites flank neomycin resistance gene. When bred with FLPe mice, neocmycin resistance gene is removed. f) LoxP sites flank exon 5. When bred with Tissue Specific Cre Mice, exon 5 is removed (g). Experimental Design Materials and Methods General Design and Strategy (Fig.3) PCR/Clonin g (Fig.3a,3b) Embryonic stem cell generation and transfection (fig.4) Screening for knockout mice (fig.4) Figure 2. Heparan Sulfate degradation pathway. Enzyme deficiencies at certain steps (shown as circle with slash) block pathway progression. Failure to acetylate the non-reducting terminal α-glucosamine residue results in MPS IIIC. N-acetylglucosamine 6-sulfatase H 2 COH O O COOH O S N S O O H 2 CO S NAc O~ O O O COOH O H 2 CO S NAc O~ H 2 COH O O COOH O S NAc O O H 2 CO S NAc O~ O O H 2 CO S NAc O~ Heparan N-Sulfatase MPS IIIA N-acetyltransferase MPS IIIC N-acetyl-  -D-glucosaminidase MPS IIIB MPS IIID  Conditional knockout mice: a means to study pathogenesis by restricting MPS IIIC phenotype to certain cell types, lineages or tissues.  Cells deficient in N-acetyltransferase:  Unable to take up enzyme from extracellular fluid or from nearby N-acetyltransferase + cells, due to membrane-bound characteristic of enzyme.  Exon 5 as candidate region for deletion:  Removal induces frameshift mutation.  Isolated exon, surrounded by ample introns.  Codes for a transmembrane domain.  Occurs relatively early in protein.  Fragment B and C Amplification: amplification currently under way.  Insert fragments B and C directly into the pBY49a vector.  Troubleshooting-Frag BC initial PCR failures:  Increase specificity of PCR reaction (raise annealing temperature, lower Taq concentration, decrease Mg 2+ concentration, lower template and primer concentrations), individual evaluation of primers, genomic template quality. Discussion Fragment A, as a PCR generated amplicon, was successfully TA cloned into pCR2.1 (data not shown). This fragment was then successfully cloned into the pBY49a vector (fig.5). Amplification of fragment B and C has been unsuccessful using the following PCR primers (Table 1). New primers are currently being evaluated. Figure 5. a) Lane 1; NotI-HpaI digestion of the pBY49a vector (1). Lane 2: Gel isolated NotI-EcoCRI digestion of fragment A insert derived from its pCR2.1 parent. b) DNA digestions (BamHI and HincII) of colonies transformed with a ligation of pBY49a and Fragment A (lanes 2-11). Successful ligations should yield a 1219 bp fragment as seen in lanes 2, 7, and 9. Table 1. PCR primers used for amplification of fragments A and BC. 64.4˚C5’-GAAACTCACTCTGTAGA- CCAGGCTGGCCTCAAAC-3’ Fragment BC- Reverse 64.4˚C5’-TTGTGGAGAGAAAGGA- AACTTTGAGGTTTGGGTT-3’ Fragment BC- Forward 68˚C5’-TTTATTGTGTTGGGAA- GAGTCAGAGTCCACGTG-3’ Fragment A- Reverse 68˚C5’-ACAACTCTACT- GCAGAATGTCTGTCCCC-3’ Fragment A- Forward Annealing Temperature Primer SequenceName Results a) bp b) References/Acknowledgements Ausseil, J., et al., An acetylated 120-kDa lysosomal transmembrane protein is absent from mucopolysaccharidosis IIIC fibroblasts: a candidate molecule for MPS IIIC. Mol Genet Metab, (1): p Fan, X., et al., Identification of the gene encoding the enzyme deficient in mucopolysaccharidosis IIIC (Sanfilippo disease type C). Am J Hum Genet, (4): p Hrebicek, M., et al., Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome). Am J Hum Genet, (5): p Neufeld, E.F., Muenzer, J., The Mucopolysaccharidoses, in The Metabolic and Molecular Bases of Inherited Disease, C.R. Scriver, Beaudet, A. L., Sly, W. S., Valle D., Editor. 2001, McGraw-Hill, Health Professions Division: New York. p No author Homolgous Recombination Method (and Knockout Mouse). Department of Biology, Davidson College, Davidson, North Carolina. Seyrantepe, V., et al. Lysosomal N-acetyltransferase deficient in mucoplysaccharidosis type IIIc is encoded by the TMEM76 gene on human chromosome 8. in 9th international Symposium on Mucopolysaccharide and related Diseases Venice, Italy. I would like to personally thank Liz Snella, Mary Jane Long, and Stephanie Patokca for all their help around the lab. Special thanks to Rafi Awedikian for training me in the techniques of cloning and Matthew Ellinwood for his endless support. This work was supported in part by grants from the National MPS Society Inc., A Life for Elisa- Sanfilippo Children’s Research Foundation, and the NSF-REU research grant. Step 1. Isolate developing embryo at blastocyst stage. This embryo is from a strain of mice with gray fur. Step 2. Remove embryonic stem cells from gray-fur blastocyst. Grow stem cells in tissue culture. Step 3. Transfect stem cells with targeting vector construct. Select for homologous recombination by growing stem cells in neomycin and gancyclvir. Step 4. Remove homologously recombined stem cells from petri dish and inject into a new blastocyst that would have only white fur. Step 5. Implant several chimeric blastocysts into pseudo-pregnant, white fur mouse. Step 6. The progeny will be normal white fur mice but others will be chimeric mice. Chimeric mice have many of their cells from the original white fur blastocyst but some of their cells will be derived from recombinant stem cells. Step 7. Mate the chimeric mice with wild-type white fur mice. If the gonads of the chimeric mice were derived from recombinant stem cells, all the offspring will have gray fur. Every cell in gray mice are heterozygous for the homologous recombination. Step 8. Mate heterozygous gray mice (+/ H) and genotpye the gray offspring. Identify homozygous recombinants (H / H) and breed them to produce a strain of mice with both alleles knocked out. The pure breeding mouse strain is a "knockout mouse". Figure 4. Schematic of ES cell recombination and knockout mouse generation (work to be conducted by the University of Iowa Gene targeting core facility). Illustration from Human Chromosome 8 Figure 1. Chromosomal location of the HGSNAT gene and predicted membrane topology for the N- acetyltransferase enzyme it codes for. Shown are the 11 trans-membrane domains of the protein (Fan et.al 2006).