Figure 5

1
A Comparative Study of Zinc Finger Nuclease
Activity Eric Hall, Laura Young, Ronnie Winfrey
Dan Voytas RET Summer Internship 2007
Abstract In the ever advancing field of gene
therapy, one challenge faced is the targeting of
specific DNA sequences for modification. One
promising response is the development of
engineered zinc finger nucleases. Zinc finger
nucleases are a tool for targeting DNA in plants,
animals, and insects. Current research in this
novel area of study has been focused towards
improving the ability to engineer modular
assembly zinc finger arrays. One obstacle is the
varying binding affinity among different zinc
finger arrays. Our research involved comparing
the effect of replacing the third finger on a
known poor-binding zinc finger array. Over the
course of our time in the lab we constructed the
zinc finger nucleases and our preliminary data
indicates that we successfully inserted the
nucleases into the vector. At this time, we are
awaiting materials to complete the gel shift
which will reveal the binding affinity of our
various nucleases.

• Methods
• Our research required two general
  processes 1) assembling the 3-finger ZFN arrays
  and 2) determining their relative effectiveness.
  Assembly of the ZFNs happens through a series of
  restriction enzyme digests and transformations
  into competent E. coli cells. Each finger is
  obtained from a library of frozen plasmids.
  These plasmids consist of a 3-4kb backbone with
  the 100 base pair ZFN sequence. To assemble a
  3-finger ZFN array, one follows this basic
  protocol (Figure 1)
• Open up the F1 vector plasmid with AgeI and BamHI
  restriction enzymes.
• Cut out the F2 finger sequence fragment using
  Xma and BamHI.
• Ligate the F2 fragment into the F1 vector using
  T4 DNA ligase.
• Repeat steps 1-3 using the new F12 vector and
  the F3 plasmid.
• After each ligation, our plasmid was transformed
  into H10B E. coli cells and grown at 37C
  overnight on LBAmp media (Figure 2).
• Next, we wanted to determine the affinity
  and specificity of our newly constructed 3-finger
  arrays. We had two choices a gel shift assay
  or a bacterial 2-hybrid assay. Because of time
  constraints we opted for the simpler gel shift
  assay.
• Preparation for the gel shift began with
  the ligation of our 3-finger fragment into the
  gel shift vector, E. coli H10B. This was done
  using the same electroporation technique as in
  previous experiments. These cells were exposed
  to isopropyl-beta-D-thiogalactopyranoside (IPTG),
  a synthetic analog of lactose which induces
  protein production using the lac operon. Once
  translation of the plasmid had begun,
  concentrations of our zinc finger protein
  increased. These proteins were then exposed to
  the target DNA sequence in the form of hairpin
  oligos and run on an agarose gel. In this
  situation, if the ZFNs bind to the target oligos,
  these larger complexes will run slower on the gel
  and be shifted when compared to the non-bound
  oligos.

Data Obtained During the course of our
experiment, many types of data were collected and
analyzed. Frequent analysis of data allowed us
to determine whether or not we should proceed to
the next step. Here is a sample of the types of
information we used DNA sequence
alignment of our ZF array fragments showed 100.0
matching between our constructed ZFN plasmids and
our desired sequence (Figure 3).
The successful growth of our transformed E.
Coli cells demonstrated adequate expression of
our desired sequence (Figure 2). The
presence of a 100-300bp band on our 0.8 agarose
gel after electrophoresis is indicative of the
successful insertion of our ZFN fragment into our
vector plasmid (Figure 4). Our gel
shift assay would have shown us whether or nor
the ZFN protein bound to our target oligo (DNA
sequence). Because of time restraints, this
assay was not completed. Figure 5 shows a sample
mobile shift assay gel.
Background A zinc finger array is a modular
assembly of three fingers of DNA each of which
code for a protein that binds to a specific three
base pair sequence on target DNA. The finger
array is bound to a zinc ion which in turn can be
fused to a restriction enzyme. When used in
conjunction with a second zinc finger/restriction
enzyme, the dimer formed can be used to pinpoint
a precise location for cutting double-stranded
DNA. It is this ability to precisely open a
location on DNA that holds promise in the field
of gene therapy. With this technology, organisms
such as plants can be modified to be more disease
resistant or have higher yields. In humans, this
technology can improve the success of gene
therapy in the treatment of diseases such as
cystic fibrosis or Parkinsons. Previous
research has been conducted by the Voytas lab
regarding the binding affinity of various G
series three finger arrays (G referring to the
first nucleotide of the target DNA). From that
research it was indicated that there was a
correlation between binding success and the type
of third finger. For our project, we set out to
construct fingers in order to compare the effect
of replacing the poor-binding third finger with a
more positive finger.
Discussion of Results Our preliminary data
showed good quality ligations of our ZFN into
vector plasmids. The presence of 100-300bp bands
on our 0.8 agarose gels represents our ligated
ZFN fragment, and these bands were present after
each ligation. Sequencing data also showed a
high quality ligation into our vector plasmids.
Due to time restraints, our gel shift assay
was not completed, but would have shown in a
qualitative sense whether or not our ZFN
protein bound to its target DNA sequence.
Figure 1 Standardized Reagents
and Protocols for Engineering Zinc Finger
Nucleases by Modular Assembly, Nature Protocols,
Vol. 1 No. 3, 2006
Figure 2 0.8 agarose gel showing
distinct 200-300bp fragments, indicating the
successful ligation of our ZFN sequences into the
vector plasmids.
Figure 4 E. Coli H10B after 15hrs growth
at 37C. Visible colonies are those which have
taken up the desired ZFN sequence and can
survive on media infused with ampicillin.
References David Wright, et. al.
Standardized Reagents and Protocols for
Engineering Zinc Finger Nucleases by Modular
Assembly. Nature Protocols. Volume 1 No.3.
1637-1652.
Research Question How does the replacement
of one finger on a known, poor-binding three
finger zinc finger nuclease affect binding
affinity?
Figure 3 An example of a DNA
sequence alignment, similar to the one we used to
determined the validity of ZFN sequences once
ligated into host E. coli cells.
Figure 5 Sample mobile shift assay gel.
The 3rd lane from the left shows a significant
shift in the protein location, representing the
proteins which were bound to the target complex.
Acknowledgements We would like to thank Ron
Winfrey for his guidance and support during our
research this summer. In addition, we would like
to thank Dan Voytas for opening his lab to us.
We also appreciate the help and patience of Jeff,
Pete and Fengli as we made our way through the
summer. Finally, thank you to the National
Science Foundation for making this internship
possible for all of us this summer.