Transcript Document

Research Experience in Molecular Biotechnology & Genomics
Summer 2007
Center for Integrated Animal Genomics
Production of a Conditional Knockout Mouse Model for the Deficient Enzyme in Mucopolyaccharidosis IIIC:
Heparin Acetyl-coenzyme A:α-glucosaminide N-acetyltransferase
Kaizan J. Kalyaniwalla1, Rafi Awedikian2, and N. Matthew Ellinwood2. 1Biochemistry, University of Wisconsin-Madison, 2Department of Animal Science, Iowa State University
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.
Step 1. Isolate
developing embryo at
blastocyst stage. This
embryo is from a strain
of mice with gray fur.
Materials and Methods
General Design
and Strategy
(Fig.3)
Embryonic
stem cell
generation and
transfection
(fig.4)
PCR/Clonin
g (Fig.3a,3b)
Screening
for knockout
mice (fig.4)
Step 3. Transfect stem
cells with targeting vector
construct. Select for
homologous recombination
by growing stem cells in
neomycin and gancyclvir.
Experimental Design
Exon 4
Exon 5
Exon 6
a)
Exon 4
Exon 5
Exon 6
X
X
PCR amplification of
three fragments covering
the region of interest
d)
Step 5. Implant
several chimeric
blastocysts into
pseudo-pregnant,
white fur mouse.
TK
LoxP
Exon 5
Neo
A
b)
B
1348 bp
C
Exon 4
Exon 5
Step 1
FRT
LoxP
LoxP
Breeding with
FLPe Mice
PGK-TK
FRT
pBY49a
7474 bp
LoxP
Exon 4
Amp
Exon 5
Exon 6
f)
pBYLoxPa
FRT
3004 bp
Breeding with
TissueSpecific Cre Mice
Exon 4
Exon 6
LoxP
Results
1
2
b)
2
3
4
5
6
7
8
9
10
11
7000
1600
H2CO S
COOH
H2COH
O
O
O
O
O~
O
NS
NAc
OS
3.10.1.1
2.3.1.78
O
O
O
N-acetyltransferase
MPS IIIC
H2CO S
COOH
H2COH
Heparan N-Sulfatase
MPS IIIA
O
O~
O
NAc
NAc
OS
3.2.1.50
N-acetyl-a-D-glucosaminidase
MPS IIIB
3.1.6.18
H2CO S
COOH
O
O
O
O~
NAc
3.2.1.31
H2CO S
O
O~
NAc
3.1.6.14
N-acetylglucosamine 6-sulfatase
MPS IIID
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.
•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.
500
1600 bp
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.
Name
Primer Sequence
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".
Annealing
Temperature
Fragment A- Forward
5’-ACAACTCTACTGCAGAATGTCTGTCCCC-3’
68˚C
Fragment A- Reverse
5’-TTTATTGTGTTGGGAAGAGTCAGAGTCCACGTG-3’
68˚C
Fragment BC- Forward
5’-TTGTGGAGAGAAAGGAAACTTTGAGGTTTGGGTT-3’
64.4˚C
Fragment BC- Reverse
5’-GAAACTCACTCTGTAGACCAGGCTGGCCTCAAAC-3’
64.4˚C
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
http://www.bio.davidson.edu/Courses/genomics/method/homolrecomb.html)
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).
a)
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.
LoxP
Amp
g)
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.
Exon 6
Neo
e)
Step 2
PGK-Neo
Homologous
Recombination
FRT
3500 bp
Step 2
Human Chromosome 8
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).
Step 4. Remove homologously
recombined stem cells from petri
dish and inject into a new
blastocyst that would have only
white fur.
c)
Step 1
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).
Step 2. Remove
embryonic stem cells
from gray-fur blastocyst.
Grow stem cells in tissue
culture.
Table 1. PCR primers used for
amplification of fragments A and BC.
Discussion
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 Mg2+ concentration, lower template and
primer concentrations), individual evaluation of
primers, genomic template quality.
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, 2006. 87(1): p. 22-31.
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.
Hrebicek, M., et al., Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome). Am J Hum Genet, 2006.
79(5): p. 807-19.
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.
No author. 2002. Homolgous Recombination Method (and Knockout Mouse).
<http://www.bio.davidson.edu/Courses/genomics/method/homolrecomb.html> 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. 2006. 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 ElisaSanfilippo Children’s Research Foundation, and the NSF-REU research grant.
Program supported by the National Science Foundation Research Experience for Undergraduates
DBI-0552371