Animal model. The normal house mouse, M. musculus, line C57bl/6 was chosen for this experiment. C57bl/6
mice have a normal phenotype and no mutations in the MECP2 gene. CRISPR/Cas9 was injected into blastocysts of C57bl/6
mice to induce insertions or deletions in the 3’UTR that resemble insertions
and deletions in the 3′ UTR of RTT patients.
CRISPR/Cas9. Clustered Regularly Interspaced Short Palindromic Repeats
and CRISPR-associated proteins (CRISPR/Cas9) edits genomes by delivering the
CAs9 nuclease and guide RNA (gRNA) complex into a cell. Spacer sequences are
transcribed into short RNA sequences that guide the system to matching
sequences of DNA. The cell’s genome is then cut at a target location when Cas9 binds
to the DNA, allowing new genes to be added or existing genes to be removed (Hsu
et al., 2014).
PCR verification of CRISPR/Cas9 insertions or deletions. Polymerase
chain reaction (PCR) makes many copies of a specific DNA region. PCR requires Taq polymerase and DNA primers designed
for the DNA region of interest. The DNA is denatured via heat and provides a
single-stranded template. Two primers are used that flank the target region and
anneal to the opposite strands of template DNA by complementary base pairing.
Primers are then extended by polymerase and the region between them is copied.
Results are analyzed with gel electrophoresis, a process in which fragments of
DNA move through an agarose gel matrix by electric current and DNA fragments
are separated according to size. DNA segments of the same length will form a
band on the gel that is made visible by ethidium bromide and UV light. PCR will
show me that I’ve transgenically modified the animal (Garibyan & Avashia,
Western Blot analysis of MeCP2 expression. Western blots determine
if modifications are causing changes in expression. This method demonstrates
different properties of proteins based on molecular weight. Mouse brain tissue
is homogenized, and then gel electrophoresis is performed on a polyacrylamide
gel, which separates the proteins by molecular mass. Proteins are then
transferred to a membrane made of polyvinylildene difluoride and stained with
antibodies to target a specific protein. Horseradish peroxidase-linked secondary
antibodies that recognize the primary antibody are then applied. The Western blot
is then analyzed by using a chemiluminescent agent, such as 4-chloronaphthol.
Size approximations are taken by comparing stained bands to the ladder bands
(Mahmood & Yang, 2012). If 3′ UTR variants cause a change in expression, I’d
see changes in the band optical density.
Sanger sequencing verification of CRISPR/Cas9 SNPs. Four sequencing
reactions are created for each nucleotide present in DNA. Deoxynucleotides
(dATP, dGTP, dCTP, or dTTP) are added to a reaction vessel. A primer strand is
bound to the template strand and a second strand assembles on top of the
template, creating double stranded DNA. Fluorescent dideoxynucleotides (ddNTPs)
are added to the reaction vessel and the chain terminates wherever a ddNTP is
randomly incorporated. The size of the fragments present in each base-specific
reaction is measured by electrophoresis, which enables separation of DNA
fragments by size and color with single-base resolution when run on a
polyacrylamide gel (Shendure et al., 2017).
Rotating rod test for assessment of motor control. Mice were placed
on a rotating rod that accelerates from 4 to 20 rotations per minute. Each
trial has a maximum duration of 600 s. The end of the trial was determined by
the mice either falling off the rod or turning with the rod two times. This
test was repeated 4 times per day for 4 days, with 10 minutes between each
trial (Deacon, 2013). Transgenic mice were found to fall off the rod more
frequently or turn with the rod multiple times, thus demonstrating that
transgenic MeCP2 mice suffer from impaired coordination. Additionally,
transgenic MeCP2 mice have abnormal forepaw movements and forelimb clasping,
which is a characteristic of neurologically impaired mice.
Open field test for assessment of anxiety-like behaviors. Mice were
placed in the center of a 40 x 40 cm open field space. Behavioral assessments
include grid line crossing and thigmotaxis, or the tendency of a mouse to
remain close to walls (Seibenhener & Wooten, 2015). Transgenic MeCP2 mice displayed
a decrease in vertical activity, and increased thigmotaxis, which is associated
with greater anxiety.