Transcript Document

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 A:alpha-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 (acetylcoenzyme 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
Exon 4
Exon 5
a)
Exon 4
Exon 5
Exon 6
c)
PCR amplification of
three fragments covering
the region of interest d)
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.
X
X
TK
LoxP
Exon 5
Neo
A
b)
B
C
Exon 4
3500 bp
e)
Step 1
Homologous
Recombination
Figure 4 c). Marker present
and a band is
Present in lane 3
FRT
1348 bp
Exon 5
Exon 6
Neo
Step 1
Step 2
FRT
LoxP
Step 2
Breeding with
FLPe Mice
PGK-Neo
LoxP
Human Chromosome 8
Exon 6
PGK-TK
FRT
LoxP
pBY49a
pBYLoxPa
7474 bp
3004 bp
Amp
Exon 4
Exon 5
Engineering a Knock-out Mouse
Exon 6
f)
FRT
LoxP
Breeding with
TissueSpecific Cre
Mice
Amp
g)
Exon 4
Exon 6
LoxP
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).
Figure 1. Chromosomal location of the HGSNAT gene
and predicted membrane topology for the Nacetyltransferase enzyme it codes for. Shown are the 11
trans-membrane domains of the protein (Fan et.al 2006).
ddss
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.
RESULTS (for some of 96 samples).
Figure 2. Pathway of enzymatic degradation of sulfoglucosaminyl
residues in heparan sulfate. Sulfamidase and alpha-Nacetylglucosaminidase 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.
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 5.) knockout mouse production. Illustation (some illustrations obmitted). from
http://linguamedica.jp/mita/20030618/knockout/knockout.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.
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 Children’s 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-CoA:alpha-glucosaminide N-acetyltransferase in skin fibroblasts. Proc Natl Acad Sci U S
A. 1978 Oct;75(10): 5188
No author. Engineering a KnockoutMouse. <http://www.fhcrc.org/science/education/courses/cancer_course/basic/approaches/elimination.html> F red Hutchinson Cancer Research
Center.
Program supported by the National Science Foundation Research Experience for Undergraduates
DBI-0552371