December 4, 2013
Protein Trapping Through Transposable P-elements
Kokila Shankar
Introduction
Protein trapping is a widely used lab technique which seeks to insert an artificial exon, through
the use of splice donors and acceptors, into an endogenous DNA sequence. As this does not disrupt
upstream and downstream splicing events (Morin et. al, 2001), it proves useful for observing cellular and
subcellular gene expression. The reason that protein trapping is effective is due to naturally occurring
transposons, and when using Drosophila, these transposons include P-elements, which can be modified
to “provide vectors for efficient DNA-mediated gene transfer” (Quniones-Coello et. al, 2007). P-element
transposons encode for the P transposase enzyme and are flanked by inverted repeat sequences of
nucleotides. It moves in the genome through a “cut-and-paste” mechanism driven by the transposase.
In this lab, the transposable P-element was modified to carry a green fluorescent protein (GFP)
coding sequence. GFP is commonly used as a molecular and cellular tag as it can be observed in unfixed,
live tissue and dynamic cell processes. When in the P-element, it is known as the reporter gene due to
its role in protein trapping – the reporter gene’s expression is used to screen for positive candidates.
Generally to insert a reporter gene into a P-element, DNA coding for transposase and a vector
containing the reporter gene are crossed, leading to random transposition of the reporter gene into the
P-element (Wikipedia, 2008).
In order to analyze the effectiveness of the protein trap, polymerase chain reaction (PCR) was
used to amplify the regions of DNA containing part of the transposon sequence through its cycles of
denaturation, annealing, and elongation. This DNA was then run through a gel to look for fragment
length and specificity. While there are many different types of PCR, two types that are used in gene trap
experiments are inverse PCR and TAIL-PCR. Inverse PCR only requires one sequence to be known to
generate a primer, which is an advantage over conventional PCR, and it is mostly useful for determining
insert locations (Wikipedia, 2012). Thermal Asymmetric Interlaced (TAIL) PCR uses a set of nested primer
pairs to generate different DNA fragment lengths of varying specificity in order to isolate unknown
sequences flanking a known region of the genome (Wikipedia, 2013).
Materials and Methods
Selecting for positive candidates: In order to initiate the protein trap, different strains of Drosophila
containing the transposase and the vector containing GFP needed to be crossed. The female
P[GA]/P[GA] was the injured P-element (inhibited expression of transposase) containing the reporter
gene and the male 2,3 Cy/Bc was the stable source of transposase. These two lines were crossed in
order to randomly transpose the GFP into the fly genome. As the offspring needed to have the 2,3
Cy/[GA] genotype to receive both components of the P-element in order for transposition to occur, only
a fraction of the total offspring were positive. A positive result was selected for based on the offspring’s
phenotype: evidence of transposition was seen through variegation in eye color instead of the white eye
color common to the wild type. These flies were then crossed with a wild type line, brooded, and
prepared for a second round of screening. This time the larvae were screened under a light microscope
with UV light to detect GFP fluorescence. Positive results expressed a concentrated region of
fluorescence compared to the overall autofluorescence seen under the microscope. Once again, these
were separated, crossed with a wild type line, and brooded to prepare for a third screening. The larvae
were again screened the same way as before; in this screen, a positive result looked like an actual
fluorescent pattern in the larvae, such as the concentration of fluorescence in the gut. After these
positive flies had been identified, they were then separated to be prepared for DNA extraction and
analysis to determine whether the GFP was actually being expressed as part of a stable line, and where
in the genome the transposon was located.
DNA extraction: Adult flies from the positive lines D1, D2, D29, and D33 were taken in different rounds
of extraction as these lines survived through the tertiary screen because they expressed fluorescence.
To extract the DNA, the flies were frozen and homogenized with a pestle in a solution containing a
buffer, chelation reagent, and detergent. After incubating and adding potassium acetate solution, the
mixture was put on ice and then centrifuged. The supernatant was removed, respun, and mixed with
isopropanol to precipitate the DNA out of solution. Finally the DNA was washed with ethanol and
dissolved in a buffer.
TAIL-PCR and gel analysis: The TAIL-PCR procedure used three sets of nested primer pairs – one primer
was complimentary to the transposon and the other was an arbitrary degenerate (AD) primer which
annealed to locations on the genome. There were three rounds of PCR performed on the DNA using
each set of primers, decreasing in size from largest fragment flanked by the primers to the smallest. For
both the P-element and GFP ends, specific primers were designed to go in the 5’ and 3’ directions. In the
TAIL 1 (first round) reaction, genomic DNA was mixed with Mg buffer, dNTPs, Taq polymerase, specific
TAIL 1 primer, AD primer, and water. This mixture was then run through the PCR machine for numerous
cycles of denaturing at 94O, annealing at 65O, and elongating at 72O. The reaction products were diluted
to a concentration of 1:50, and that solution was used in the TAIL 2 reaction, which followed the same
procedure except with the substitution of the nested specific TAIL 2 primer for the TAIL 1 primer.
Additionally, the length of time the mixture spent in the PCR machine was shorter, and annealing took
place at 64O. For the third round of the reaction, the same procedure was followed, the only changes
being the substitution of the nested specific TAIL 3 primer instead of TAIL 2 and the reduction of the
annealing temperature to 44O.
The amplified PCR fragments that resulted from the TAIL-PCR reactions were then run through a 1.5%
agarose gel at around 110V in order to analyze the fragments’ length and specificity. Samples of DNA
were taken from each round of the TAIL-PCR reaction and run through the gel.
Sequencing DNA from PCR analysis: DNA from the TAIL 3 reaction which produced a small, specific band
on the gel was then run through a large scale PCR gel to produce one large band of DNA. The specific
band was then cut out of the gel and run through a DNA extraction procedure to generate a large
quantity of DNA, which could then be sent in for sequencing.
The sequencing results produced a nucleotide sequence. This was then put into a BLAST search
to find out what specific gene was trapped. The resulting gene name was then entered into a FlyBase
search in order to obtain the location of the gene in the genome. Based on the orientation of the gene,
it was determined whether the gene was on the top or bottom strand. Given the direction of the Pelement, fusion could only exist if the gene direction and P-element direction were the same. If fusion
was possible, then a decorated FastA was generated in FlyBase and the P-element sequence was
searched for within the genome region in order to find out whether the insert site was in an intron. if
the insert site was in an intron, then both the upstream exon and the FastA from FlyBase were
translated and a search was performed to discover the exact fusion site of the gene by looking for the
exon’s amino acid sequence within the entire protein sequence.
Results
PCR gel analysis:
Figure 1a – Gel of various fly lines which passed the tertiary round of screening. Each line has DNA from
each round of TAIL-PCR for both 5’ and 3’ primers.
Figure 1b – inset of D1 results
Figure 1c – inset of T8 results
Figure 2 – Preparative gel showing DNA from two positive lines in single large bands in preparation of
undergoing DNA extraction
Sequence analysis:
Line 5’ D1 found the trapped gene to be nicotinic acetylcholine receptor α 7E.
Line 3’ T8 found the trapped gene to be gilgamesh.
Discussion
The results from the PCR showed a few promising inserts, which were then sequenced to see if
they were genuine trapped genes or not. Two of the lines which tested positive were D1 using the 5’ Pelement primer and T8 using the 3’ P-element primer. This can be seen through the alignment of the
bands on the gel. When looking at a TAIL-PCR gel, each round should produce a specific result. In round
1, since the primer pair is not very specific, there should be a lot of bands of varying lengths. Round 2
should have fewer bands of lengths that are closer together in size since the primer is more specific. By
round 3, there should be one concentrated band since the last set of primers is the most specific and
should be able to generate a large quantity of similarly cut DNA fragments. Additionally, as each set of
primers gets more specific, the fragment size decreases, so the highly concentrated band from the
round 3 well should be lower on the gel than the more concentrated bands from the round 2 well.
Furthermore, since the P-element can only be going in the 5’ or the 3’ direction, a positive result will
only appear on one of the two halves of the gel. This can be seen in line D1 (Figure 1b) as the DNA
fragments increase in specificity and decrease in length on the 5’ side over the three rounds of the TAILPCR reaction. Similar results can be seen in the 3’ P-element side of the T8 line. While this is an example
of a successful TAIL-PCR gel, there are also failed gels, and they are not successful for a few different
reasons. First, there were no specific bands produced, as seen in lines T3 and T6. Although it looks like
the first set of primers were able to generate a couple of specific bands, it was not enough to continue
into the subsequent rounds of the reaction. Another example of a failed result can be seen in lines D3
and T7: in this case, the band from the round 3 well is higher than the band from the round 2 well,
signifying that the more specific primer actually produced a longer, and therefore less specific, DNA
fragment. Since that result does not make sense in regards to the TAIL-PCR procedure, it needs to be
disregarded. These are some of the problems that can occur when using TAIL due to the fact that only
one set of the nested primers are known, which means that a match is not always guaranteed as
opposed to inverse PCR, where only one known primer is used. TAIL-PCR as a whole, even though it can
produce some failed results, has advantages over inverse PCR, the other method considered in this lab.
Since TAIL uses more sets of primers than inverse (inverse PCR just uses one primer), there is increased
specificity, which could help to better pinpoint the sequence. Also, inverse PCR involves breaking up the
DNA into multiple restriction fragments through restriction enzymes and joining them together in a
circle with ligate; that process as a whole poses a greater risk of complication and there are probably
more ways for the DNA to be damaged.
Line D1 came up with nicotinic acetylcholine receptor α 7E as the trapped gene. While it was
from the 5’ P-element end, its orientation was on the bottom strand. Since the P-element and the gene
were going in opposite directions, fusion to the genome was not possible, and it was not necessary to
consider the gene being in frame. This analysis actually did not support the conclusion made about the
line after the PCR was run, since the gel produced positive results. This nicotinic acetylcholine receptor
serves as a neurotransmitter gated-ion channel, so it would most likely be found in the cell embedded in
the cell membrane as a transmembrane protein. Primarily these would be seen in neurons, as they help
to regulate electrical signals within the body. Its main function is regulating cation transport across the
cell membrane.
Line T8 came up with gilgamesh as the trapped gene. This gene was from the 3’ P-element end,
but both the GFP-open reading frame and the gene had the same orientation, so fusion was possible.
However, the gene did not turn out to be in frame with the surrounding genome region, so this line also
did not turn out to be a positive match. Again, this failed to support the conclusions drawn after the
PCR/gel analysis. The gilgamesh gene serves a wide variety of functions in the Drosophila body. Its main
molecular functions are in ATP binding and serine/threonine kinase activity. This leads to regulation of
activities like glial cell migration, olfactory learning, spermatogenesis, and protein phosphorylation. As a
result, these proteins are related to both the nucleus and the cell membrane, and are probably found in
a wide variety of tissues, one example being glial cells in the eyes (Hummer et. al, 2002).
Conclusion
Overall, this lab was designed to test a GFP-fusion protein trap to study the way transposable Pelements functioned in Drosophila. Through the use of techniques including genetic screening, DNA
extraction, and TAIL-PCR, the flies’ genomes were consistently pinpointed to find a specific, stable
region of the P-element expressing GFP. However, the sequences thought to be positive results actually
did not match the conditions necessary for proper fusion and insertion.
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Cell Migration in the Drosophila Eye Requires Gilgamesh, Hedgehog, and Eye Specification Genes."
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