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					                                                                       synthesis are involved in long-term memory acquisition and
     Blocking Homer 1c                                                 transcriptional activation has been altered in both learning-
                                                                       impaired and unimpaired rats during long-term potentiation
  expression with siRNA in                                             (Lanahan, Lyford, Stevenson, Worley, & Barnes, 1997).
 HEK 293 cells as a Neuronal                                           Several studies have observed changes in transcriptional
                                                                       regulation of individual genes in aged-related memory
  ModelCharacterization of                                             impairment, including genes involved in transduction
                                                                       pathways, synaptic transmission, and synthesis of excitatory
 siRNA’s against Homer 1c;                                             amino acids (Adams et al., 2001; Clark et al., 1992; Colombo
                                                                       & Gallagher, 1998; Colombo, Westel & Gallagher, 1997;
   Aa Ggene iInvolved in                                                                                                               Formatted: Font: (Default) Times, 10 pt
  Llearning and Mmemory                                                                                                                Formatted: Centered


          Camille N. Camacho Rodríguez
         Universidad del Este, Carolina P.R.

    Corinna Burger, PhD & Hilary Gerstein,
           Department of Neurology
                      &y
Hilary Gerstein, Neuroscience Training Program
UW-MadisonSchool of Medicine and Public Health

Abstract                                                                                                                               Formatted: Font: 10 pt
                                                                                                                                       Formatted: Indent: First line: 0"
It i’s believed that Homer 1c proteins have an important role          Nicolle, Colombo, Gallagher, & McKinney, 1999; Smith,           Formatted: English (United States)
in learning and memory since they may regulate Group 1                 Adams, Gallagher, Morrison, & Rapp, 2000; Stenvers, Lund,
Metabotropic Glutamate receptors. However, the exact role of           & Gallagher, 1996).                                             Formatted: English (United States)
Homer 1c protein is unknown. The purpose of this study is to                                                                           Formatted: Font: 10 pt
design and test the efficacy of three siRNA sequences that                                                                             Formatted: Indent: First line: 0"
could induce knockdown of Rat Homer 1c in vitro using                  The Homer 1c gene appears to be upregulatedup-regulated in
HEK 293 cells. After characterization, effective siRNA                 aged rats with superior memory, therefore Homer 1c may play     Formatted: Font: Italic, English (United
                                                                                                                                       States)
sequences would be cloned in to AAV vector for further use             a role in enhanced memory formation. Homer proteins are
inbehavioralin behavioral tests. We amplified the shRNA                found in the post-synaptic density of synapses and have been    Formatted: English (United States)
constructs and transfected them into HEK 293 cells, after a 48         shown to regulate Group 1 metabotropic glutamate receptors      Formatted: English (United States)
hour incubation, protein was extracted and quantitated with            (G1 mGluRs). However, it is unclear whether Homer 1c is
                                                                                                                                       Formatted: English (United States)
Westernwith Western Blot. Preliminary data suggests that two           important in cognition.
of the three siRNA’s could be effective in down regulating                                                                             Formatted: English (United States)
gene expression of Homer 1c.                                           siRNA (small inhibitory RNA) is a tool used to selectively      Formatted: Font: 10 pt
                                                                       down-regulate targeted proteins. TIn this study tested the
                                                                                                                                       Formatted: Indent: First line: 0"
                                                                       efficiency of previously designed Homer 1c siRNA’s in a
                                                                       non- neuronal cell type (HEK 293) to see if protein products    Formatted: Font: 10 pt, Not Bold, English
                                                                       decreased. Since Homer 1c appears to be up-regulated in         (United States)
Introduction                                                           enhanced learners, we could study Homer 1c’s role in learning   Formatted: Font: 10 pt
                                                                       and memory by knocking down gene expression and looking         Formatted: Font: 10 pt
                                                                       for changes in cognition.
Hippocampal circuits are engaged in consolidating explicit                                                                             Formatted: Font: 10 pt
memories (declarative memories), such as the conscious                                                                                 Formatted: Font: 10 pt
recollection of facts, and forming spatial memories.                    We designed short hairpin RNA (shRNA) cassettes
Microarray analysis identified several genes that were                                                                                 Formatted: Font: 10 pt
                                                                       containing the siRNA’s sequences that bind to Homer 1c’s
transcribed differently in aged enhanced learners versus the           mRNA specifically, cleave it thereby preventing gene            Formatted: Font: 10 pt
aged learning impaired rats in the hippocampus (Burger et al.,         expression and subsequent protein expression. After             Formatted: Font: 10 pt
2007). These genes might play an important role in memory              transfecting the cells, there was a 24-48 hour incubation       Formatted: Indent: First line: 0"
acquisition in aged rats. It is known that mRNA and protein            period, then we performed Western Blotting technique to
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                                                                 133
assess the presence of Homer 1c protein in cells. To analyze                     After the 48 hour incubation period, we removed the
the data we will use Image J software. Image J is a semi-               media from cells and scraped them with cold PBS (using cold
quantitative analysis tool that estimates the percentage of             PBS helps us optimize cell detaching). We collected the cells
protein present in cells provided by the NIH.                           with a microcentrifugemicro centrifuge at top speed
                                                                        (14,000rpm). Later, the cells were resuspended in freeze-thaw
                                                                        lysis buffer (40l per 6cm dish). After the resuspension, tubes
                                                                        were frozen in dry ice. This cycle was performed two more
Materials & Methods                                                     times. Then, we thawed the cell lysate and added 250 l of
                                                                        Benzonase to digest DNA. After mixing the sample, an
2.1 shRNA Ccassette Ssynthesis                                                                                                            Formatted: Font: Helvetica, 12 pt, Bold
                                                                        incubation period of 10 minutes at room temperature followed.
         After the sequence of the Homer 1c gene was known,             Then, we measured protein concentration using the Bradford
a lab memebermember used BLAST to design 19 bp long                     Assay. Then, an equal volume of SDS 2X buffer was added.
antisense sequences. The sense and antisense sequences were             Later, the samples were heated at 95C for 10 minutes.
reconstituted in a hairpin construct containing a RNA Pol III           Immediately after the heating phase, the samples were loaded
Promoter Region. Oligos were ordered from Origene. The                  in a 12% Polyiacrylamide gel
vector was previously designed by a lab member and it was
designed using NTI Software (Invitrogen).                               and analyzed by SDS-PAGE. These samples must be loaded
                                                                        while still warm to prevent precipitation of the SDS with
                                                                        Laemmli buffer.
2.2 AmplifiyngAmplifying                    the     shRNA                                                                                 Formatted: Font: Helvetica, 12 pt, Bold
cCassette.
          We amplified our three shRNA cassettes (Homer 1c              2.6 Western Blotting                                              Formatted: Font: Helvetica, 12 pt, Bold
Rat #475, Homer 1c specific and Homer 1c Rat #1012) with                         12% Tris-HCL (BioRad Ready Gel) was used. 100μg
PCR. Homer 1c Rat #1012 was amplified according to the                  of protein wasere loaded per well. It was runned at 130V.
Mirus siXpress Human H1 PCR Vector system protocol. We                  Semi-Dry Transfer was performed by placing the gel adjacent
sartedstarted with a Hot Start at 95°C for 3 minutes, and then          to Nitrocellulose paper in a transfer sandwich and running
                                                                                                                                          Formatted: Font: 9 pt               spcfc
                                                                                                                                                                     #475 H1c#1012
30 cycles of 95°C for 30 sec, 62°C for 20 sec, 68°C for 30 sec,         0.08A (with constant voltage) through the sandwich apparatus
with a final elongation for 10 min. Homer 1c Rat #475 was               for 90mins. After the Semi-Dry transfer was performed, we         Formatted: Font: 9 pt
aplifiedamplified with a lower anealingannealing temperature            rinsed the blot with 1X TBS+Tween for 5 minutes and               Formatted: Font: 9 pt
of 60°C. We tried to amplify Homer 1c specific oligo                    blocked with 5% milk+TBS-T for 1 hour. After blocking, we
unsuccesfullyunsuccessfully with different protocols (Cheng             incubated the blot overnight at 4°C in 1° antibody (1:200,
et al., 2006).                                                          goat/anti-Homer 1b/c/Santa Cruz). The next day, the 1°
                                                                        antibody was removed and the blot was rinsed 5x for 5
2.3 DNA Purification                                                    minutes in 1XTBS-T (10mls per wash) on a shaker. Then,            Formatted: Font: Helvetica, 12 pt, Bold
                                                                                                                                                                                100bp
        After running the PCR products in 2% agarose gel,               10mls of fresh 2° antibody (1:1000, donkey/anti-goat/Santa        Formatted: Font: 9 pt
we extracted the band. We used the Promega Wizard® SV Gel               Cruz) with the blocking solution (5% Milk+1X TBST) were
Clean-Up° kit to purify the DNA. After using the cleanup kit,           added and the blot was incubated for 90 minutes at room
we precipitated the DNA to obtain a higher yield and and then           temperature. After incubation, the blot was rinsed 5x for 5
we stored it in -20˚C for further use.                                  minutes each with 1X TBST. -After rinsing, we proceeded to
                                                                        prepare the blot for the imaging step.
2.4 Transfecting HEK 293 Ccells                                                                                                           Formatted: Font: Helvetica, 12 pt, Bold
         Approximately 24 hours prior to transfection, cells            For the imaging step we used ECL kit (Pierce). Protein
were plated to a 35% density so that they would be about 70%            densitometry was calculated by taking a digital image of the
confluent the next day. We added 4l of the TransIT-Oligo              blot and analyzed measuring the mean gray value (intensity of
Reagent (Mirus) to a sterile tube with 600l of serum-free              the bands) with Image J software (NIH).
medium. Followed by a 20 minute incubation period, we                                                                                     Formatted: Font: 11 pt
added 16.4 l of the desiganteddesignated oligonucleotide.              .
Then another 20 minute incubation period followed. After the
incubation          period,           the           TransIT-
OligoReagent/oligonucleotide complex mixture was added                  Results
dropwisedrop wise to the plated cells. After swirling the cells
                                                                                                                                          Formatted: Font: Helvetica, 12 pt
for a few seconds, we incubated the cells for 48 hours at 37°C.         3.1 Amplifying the shRNA Ccassettes
                                                                                                                                          Formatted: Indent: First line: 0"
                                                                        Since lowering the annealing temperature didn’t work with the
2.5 Protein Extraction                                                  H1c specific shRNA sequence, we tried a new protocol              Formatted: Font: Helvetica, 12 pt, Bold
                                                                                                                                          Formatted: Wrap Around
                                                                  134
specially for shRNA’s (Cheng et al., 2006). For this process                                                                                             Formatted: Font: (Default) Times, 10 pt
refer to Figures 1-6.                                                                                                                                    Formatted: Indent: Left: 0", Hanging: 0.19"
                                                                                                                                                         Formatted: Font: 9 pt
                           Mirus PCR Protocol                                                                                                            Formatted: Font: (Default) Times, 10 pt




                                                                                    Temp (C)
               105
                           95
                95
    Temp (C)




                85
                75                                             68
                65                              62
                55
                     180   200     220    240            260        280
                                    Time (s)

Figure 1. PCR using Standard Protocol (Mirus)                                   Figure 1. PCR using standard protocol (Mirus)
This protocol was used to amplify the shRNA cassettes.                          Western Blotting-label each lane of the blot so we know what protein     Formatted: Font: 9 pt
                                                                                  was probed for. Label any molecular weights that are important.
                                                                                  Label control ladder etc. In the figure legend, be sure to state the   Formatted: Font: Times, 8 pt
                                                                                  result-x was different from yThis protocol was used to amplify
                                                                                  the shRNA cassettes.



                                                                                Figure 2. Bar graph with % of densitometry semi-quantitative
                                                                                    analysisPCR product from Mirus protocol
                                                                                                                                                         Formatted: Indent: Left: 0", Hanging: 0.19"
                                                                                The presence of Homer 1c protein following different siRNA
                                                                                sequences. Maximal protein knockdown occurs with sequence #5.

                                                                                                                                                         Formatted: Space After: 6 pt
                                                                                                        Mirus PCR Protocol (Δ° C )                       Formatted: Font: 10 pt




                                                                                                105         95
                                                                                                 95
                                                                                   Temp (° C)




                                                                                                 85
                                                                                                 75                                     68
                                                                                                 65
                                                                                                 55
                                                                                                 45
                                                                                                 35                                                                              40
                                                                                                      180            230                     280
                                                                                                                   Time(s)

                                                                                                                                                         Formatted: Font: 9 pt
                                                                                                                                                         Formatted: Font: 9 pt
  100b                                                                                                                                                   Formatted: Font: 9 pt
                                                                                                                                                         Formatted: Font: 9 pt
                          spcfc #101
                 #475 H1cH1c
                   #47           #1012
                                                                                                                                                         Formatted: Font: 9 pt
                   5     spcfc 2
                                                                                                                                                         Formatted: Font: 9 pt
                                                                                                                                                         Formatted: Font: 9 pt
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                                                                          135
     Figure 3. Western Blot w/ Desitometry AnalisysSecond PCR Protocol
     with Δ°C
     Lane containing siRNA aganist Homer1c is significantly reduced compared to
                                                                                                                     H1c spfc PRC Protocol
     controls. (P-value).A second PCR Protocol was performed with different
     annealing                                                     temperatures.
                                                                                                         105
                                                                                                                       95




                                                                                             Temp (°C)
                                                                                                          95                                                    Formatted: Font: (Default) Times, 8 pt
                                                                                                          85
                                                                                                                                      75
                                                                                                          75                            50°C       72
                                                                                                                     17x                                        Formatted: Font: 8 pt
                                                                                                          65                                                    Formatted: Font: 8 pt
                                                                                                               180          200         220          240
                                                                                                                                                                Formatted: Font: 9 pt
                                                                                                                                  Time (s)
                                                                                                                                                                Formatted: Font: 9 pt



                                                                                                                     H1c spfc PRC Protocol
                                                                                                         100
                                                                                                                       95
                                                                                                          95
                                                                                                          90
                                                                                             Temp (°C)




                                                                                                          85
                                                                                                          80                          75
                                                                                                          75                                       72
                                                                                                          70
                                                                                                          65
                                                                                                               180          200         220          240
                                                                                                                                  Time (s)

                                                                                         Figure 5. Third PCR Protocol                                           Formatted: Font: Bold
                                                                                         Protocol specially designed for shRNA’s from BMC Biotechnology.
0x
                                                                                                                                                                Formatted: Font: 10 pt, Not Bold
                100bp       40°C        50°C         60°C       70°C                                                                                            Formatted: Font: 8 pt
                                                                                                                                                                Formatted: Font: 8 pt
                                                                                                                                                                Formatted: Font: 8 pt
     Figure 4. Second PCR Product
     After the second PCR trial, only sequence #475 amplified as we Lowered                                                                                     Formatted: Font: 8 pt
     annealing temperature. Seems that the best temperature to amplify this
     sequence it’s 50°C.                                                                                                                                        Formatted: Font: 8 pt
                                                                                                                                                                Formatted: Font: 8 pt
              Since lowering the annealing temperature didn’t
                                                                                                                                                                Formatted: Font: 8 pt
     work with the H1c specific shRNA sequence, we tried a new
     protocol specially for shRNA’s (Cheng et al., 2006)                                                                                                        Formatted: Font: 8 pt
                                                                                                                                                                Formatted: Font: 8 pt
                                                                                                                                                                Formatted: Font: Bold
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                                                                                                                                                                Formatted: Justified
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                                                                                                                                                                Formatted: Font: 8 pt

                                                                                         Figure 6. Third PCR Product with Δ°C                                   Formatted: Font: 8 pt, Not Bold
                                                                                         The shRNA protocol did not successfully amplify theied H1c sequence.
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                                                                                   136
                                                                        Dr. Ana T. Mendez Merced, PhD, Universidad del Este, Carolina
                                                                        P.R.                                                               Comment [hmd1]: Because you did this
                                                                                                                                           particular work with the burger lab, it looks odd to
Discussion                                                                                                                                 acknowledge Ana first.

                                                                        Funding                                                            Formatted: Font: 11 pt
The preliminary results show that the amplified sequences
may be successful in knocking down Homer 1c gene                        IBS-SRP supported this student with funding from the:
expression. shRNA’s were difficult to amplify, the first
                                                                             Genentech Foundation #080455                                 Formatted: List Paragraph, Bulleted + Level: 1
sequence (#1012) was amplified but showed a double band.                                                                                   + Aligned at: 0.25" + Indent at: 0.5"
                                                                             National Science Foundation (DBI-0552806)
This may be because shRNA’s may interact and form
polindromic structures that causes this phenomenon                           University of Wisconsin-Madison Graduate School              Formatted: Bullets and Numbering
(Fig.Figure 2) For the second PCR (Fig.Figure 3), we made a             This research was supported by the:
temperature gradient ranging from 40°C-70°C for the other                    National Science Foundation (144 PE44)
samples (#475, H1c). We amplified shRNA #475 and it                          University of Wisconsin-Madison Graduate School              Formatted: Bullets and Numbering
seems that the best annealing temperature for this sequence
would be 50°C. After the second PCR, we used a different
protocol (Fig.Figure 5) especially for shRNA’s with no                  References
success but in this reaction we noted that primer
concentrations were high. Another PCR was performed                         1.   Burger, C, et al (2007).Changes in Transcription          Formatted: Bullets and Numbering
diluting the primer 20x. Unsuccessfully, the primer levels                       within the CA1 field of the hippocampus are
were still high and our sequence did not amplify. After this,                    associated with age-related spatial learning
we thought that the upstream promoter was interacting with                       impairments. Neurobiol. of Learning and Memory. 87,
the downstream primer and we did another PCR adding the                          21-41.Office 2004 Test Drive User Page137
                                                                                          9/1/2012.
upstream promoter after 10 cycles. The, H1c sequence did not
amplify and we still had high primer levels. We did another
PCR with the diluted primer and with a temperature gradient
                                                                            2.   Blalock, EM (2003).Gene microarrays in hippocampal
(40°C-70°C). After this PCR, we noted that the increase in
                                                                                 aging: statistical profiling identifies novel processes
temperature would also increases primer concentrations. After                    correlated with cognitive impairment.. J. of
several trials, the amplification of this sequence (H1c) was                     Neuroscience. 23(9), 3807.
unsuccessful and it i’s still a work in progress. For the
transfection,          TransIT-OligoReagent/oligonucleotide
complex mixture was added drop wise to cells before a                       3.   Klugmann, M (2005).AAV-mediated hippocampal
incubation period of 48 hours. After the incubation period, the                  expression of short and long Homer 1 proteins
protein was extracted and measured using Western Blot to                         differentially affect cognition and seizure activity in
assess the presence of Homer 1c proteins in the cells. Due to                    adult rats.. Mol and Cell. Neuroscience. 28(2), 347-
technical difficulties, the blots did not accurately confirmed                   60.
the presence of Homer proteins in the cells but a possible
knockdown occurred. After the Western Blot analysis, we
proceeded to clone the AAV viral vector with the amplified                  4.   Foa, L (2009).Developmental roles for Homer: more
templates.                                                                       than just a pretty scaffold.. J. of Neurochem. 108(1),
                                                                                 1-10.


                                                                                                                                           Formatted: Font: 11 pt
                                                                            5.   Lanahan, A., Lyford, G., Stevenson, G. S., Worley,
                                                                                 P.F., & Barnes, C.A. (1997). Selective alteration of
                                                                                 long-term potentiation induced induced trancriptional
                                                                                 response in hippocampus of aged, memory-impaired
                                                                                 rats. Journal of Neuroscience, 17, 2876-2885.


Acknowledgments                                                             6.   Cheng, D (2006).PCR-based generation of shRNA
                                                                                 libraries from cDNAs. BMC Biotechnology ,
Hilary Girstein, Neuroscience Training Program, UW Madison                       6:28(10.1186/1472-6750-6-28), 1-11.
Dr. Corinna Burger, PhD, UW Madisonn                                    Burger, C, et al (2007).Changes in Transcription within the        Formatted: Indent: Left: 0"
Sue Osting, UW Madison Department of Neurology                          CA1 field of the hippocampus are associated with age-related
Jenny Dahlberg, Neuroscience Training Program                           spatial learning impairments. Neurobiol. of Learning and
Steve Johnson, Neuroscience Training Program                            Memory. 87, 21-41.Office 2004 Test Drive User
                                                                                 Page107         10/13/099/11/0907/31/09.                  Formatted: Wrap Around
                                                                  137
Blalock, EM (2003).Gene microarrays in hippocampal aging:
statistical profiling identifies novel processes correlated with
cognitive impairment.. J. of Neuroscience. 23(9), 3807.


Klugmann, M (2005).AAV-mediated hippocampal expression of
short and long Homer 1 proteins differentially affect cognition
and seizure activity in adult rats.. Mol and Cell. Neuroscience.
28(2), 347-60.


Foa, L (2009).Developmental roles for Homer: more than just a
pretty scaffold.. J. of Neurochem. 108(1), 1-10.


    Lanahan, A., Lyford, G., Stevenson, G. S., Worley, P.F., &           Formatted: Indent: Left: 0", Tab stops:
    Barnes, C.A. (1997). Selective alteration of long-term               0.25", List tab + Not at 0.5"
    potentiation induced induced trancriptional response in
    hippocampus of aged, memory-impaired rats. Journal of
    Neuroscience, 17, 2876-2885.                                         Formatted: Font: Not Italic
                                                                         Formatted: Font: Spanish (Mexico)
                                                                         Formatted: Indent: Left: -0.25", Numbered +
    Cheng, D (2006).PCR-based generation of shRNA                        Level: 1 + Numbering Style: 1, 2, 3, … + Start
    libraries  from   cDNAs.     BMC    Biotechnology ,                  at: 1 + Alignment: Left + Aligned at: 0.25" +
    6:28(10.1186/1472-6750-6-28), 1-11.                                  Tab after: 0.5" + Indent at: 0.5", Tab stops:
                                                                         0.25", List tab + Not at 0.5"
                                                                         Formatted: Bullets and Numbering
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                                                                         Formatted: Numbered + Level: 1 +
                                                                         Numbering Style: 1, 2, 3, … + Start at: 1 +
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