MATERIALS AND METHOD

1
Research Experience in Molecular Biotechnology
Genomics Summer 2008
Center for Integrated Animal Genomics
Production of a Conditional Mouse Model for the
Deficient Enzyme in Mucopolysaccharidosis
IIIC Acetyl-coenzyme Aalpha-glucosaminide
N-acetyltransferase
Daniel B. Tata,1 Ashley Dierenfeld,2 and N.
Matthew Ellinwood,2 Biology, Bowie State
University, Department of Animal Science, Iowa
State University.

• INTRODUCTION
• The Mucopolysaccharidoses (MPSs) are a group of
  genetic lysosomal storage disorders caused by the
  accumulation of glycosaminoglycans (GAGs).
• GAG accumulation is due to insufficient enzyme
  activity which is inherited as a genetically
  determined loss of specific enzyme activity. In
  the long run, GAG accumulation leads to organ
  and tissue damage affecting primarily bones
  and/or the central nervous system.
• 7 known distinct forms of MPS exist, and are
  caused by deficiency of 11 known enzymes.
• MPS III has four known subtypes, each caused by
  the deficiency of one of four known enzymes that
  degrade the GAG heparan sulphate.
• MPS IIIC is caused by autosomal recessive
  inheritance of deficiency of the lysosomal enzyme
  HSGNAT, with combined membrane transporter and
  acetyl transfersase activty (acetyl-coenzyme A
  alpha glucosaminide N-acetyl tranferase)
• To date, no animal models of MPS IIIC exists for
  pre-clinical testing of therapy and to study
  pathogenesis.

Experimental Design
Figure 4 b) Lane 1 is the marker. Lanes 4, 5, 6,
8, 9, 10, 14, 15, 16, 17, 18, and 20 all have
bands. Figure 4 c). Marker present and
a band is Present in lane 3
Engineering a Knock-out Mouse
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).

• MATERIALS AND METHOD
• Previously the gene targeting plasmid (figure 3)
  was used to generate stable transfected mouse
  embryonic stem cells. Resultant clones that
  survived positve (NeoR), and negative (TK)
  selection were screened for appropriate
  homologous recombination by PCR screening.
• Screening was conducted in a 96 well plate format
  and samples were screened to find out if they
  could serve as potential clones to generate a
  knockout mouse (figure 5).
• PCR Reagents 2.5mM deoxyribonucleotide
  triphosphate (dNTP), 5X buffer (to maintain the
  pH), Promega taq (for primer extension), primers
  ( for DNA amplification).
• Primers used primers 13, 14, 15, 16, 35 and 36.
  13, 14, 15 and 16 are designed to amplify the
  neomycin insert, the fifth exon, and the
  construct/genomic borders.
• Polymerase Chain reaction (PCRs) A PCR was ran
  with the first set of primers (13, 14, 15 and
  16). By means of an agarose gel electrophoresis ,
  these samples were analyzed.
• Agarose Gel Electrophoresis technique used to
  separate by size, DNA fragments by means of an
  electric current applied to the gel matrix.
• SYBR Gold is a fluorescent intercalating dye.
  Low concentrations of SYBR Gold stains were
  introduced into the gel thereby facilitating the
  location of DNA during examination under U.V
  light.
• Samples that contained the neomycin insert
  produced bands when observed under u.v light.
  These were screened with primers 35 and 36 (
  these primers are designed to amplify exon 5) and
  observed after electrophoresis.

ddss
Figure 5.) knockout mouse production.
Illustation (some illustrations obmitted). from
http//linguamedica.jp/mita/20030618/knockout/knoc
kout.htm

• DISCUSSION
• Bands observed in figure 4a) and 4b) were
  produced by clones that contain the neomycin
  insert. These clones were further screened with
  primers 35 and 36 and results seen in figure
  4c.). Positive controls were as expected, which
  indicated the PCR worked. If no bands were
  observed from clones it implies no amplification
  of desired region, which is likely the result of
  no homologous recombination having taken place.
• This result is unanticipated and may indicate
  that the construct as designed did not yield any
  homologous recombinant clones. Redesign may
  employ larger arms on the construct.

RESULTS (for some of 96 samples).

Acknowledgements and References Acknowledgements
are given to the current and former members of
the Ellinwood Laboratory for their help and
assistance. Specifically Kazan Kallianawalla for
supplying graphics, Rafi Awedikian, and Ashley
Dierenfeld for assistence with experiements. For
funding support acknowledgements are give to the
Sanfilippo Childrens Research Foundation for
grant suport (http//www.alifeforelisa.org/). Fan,
X., et al., Identification of the gene encoding
the enzyme deficient in mucopolysaccharidosis
IIIC (Sanfilippo disease type C). Am J Hum Genet,
2006. 79(4) p. 738-44. 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. 3421-3452. Klein U,
Kresse H, von Figura K. Sanfilippo syndrome type
C deficiency of acetyl-CoAalpha-glucosaminide
N-acetyltransferase in skin fibroblasts. Proc
Natl Acad Sci U S A. 1978 Oct75(10) 5188 No
author. Engineering a KnockoutMouse.
lthttp//www.fhcrc.org/science/education/courses/ca
ncer_course/basic/approaches/elimination.htmlgt F
red Hutchinson Cancer Research Center.
Figure 4a) Lane 2, 3, and 4 are the
controls. Positive bands visible in lanes
10,14,15,17,and 18. Lane 6 is a ladder and
provides information on amplification size.
Figure 2. Pathway of enzymatic degradation of
sulfoglucosaminyl residues in heparan sulfate.
Sulfamidase and alpha-N-acetylglucosaminidase
are deficient in Sanfilippo syndrome A and B
respectively. As mentioned in the introduction,
acetyl-CoA alpha-glucosaminide
N-acetyltransferase is deficient in Sanfilippo
Syndrome C.
Program supported by the National Science
Foundation Research Experience for
Undergraduates DBI-0552371