|
|
| The Case: |
Mixed Signals |
|
You and your lab are researching different gene therapy treatments in hopes of finding a cure for sickle cell anemia. To test your therapies, you have been breeding in your laboratory a strain of transgenic mice, which exclusively express human sickle hemoglobin. These homozygous, transgenic mice contain no murine b-globin genes, just human b-globin genes, as shown in the electrophoresis gel in Figure 1. This colony has been carefully controlled and a hemoglobin gel is ran on each mouse to ensure that they are in fact homozygotes (i.e. expression only human sickle hemoglobin).
In order to determine if your gene therapies are having a negative effect on other genes, you decide to run MURINE cDNA microarrays on each mouse before and after they received a gene therapy treatment. These cDNA microarrays contain probes consisting of 10, 50 bp, oglionuecleotide segments from within a gene to help you determine whether or not a particular gene is being expressed (Figure 2). This is repeated for every gene in the murine genome, giving you the ability to determine the effect of the gene therapy treatment on gene expression of every gene.
You have been working on a new gene therapy that you are convinced will work. One day, one of your students comes into your office all looking all confused. “When I run the cDNA microarrays on the mice that received the new gene therapy treatment, the cDNA microarrays shows that the mice are now expressing murine b-globin. However, when I run a hemoglobin gel, it shows that the mice still express human sickle b-globin? I don’t understand what’s going on?” 
|
|
|
Case Analysis
What happened?
What do I do to ensure I won’t get conflicting data like this ever again?
Sequence of murine b-globin gene
Sequence of human sickle b-globin gene
|
Option: Include a know / need to know chart like the one below:
|
What do you know?
|
What do you need to know?
|
| |
|
|
|
Learning Goals
Goal(s)
•Learn how to access and search databases for DNA and protein sequences, and how to align and manipulate these sequences.
•Learn how to view 3-dimensional structures of protein molecules using the previously found sequences.
•Learn how to use NCBI database, BLAST, or Biology Workbench software.
•Learn about homology.
•Better understand the limitations of cDNA microarrays, gene therapy, and other state-of-the-art technologies.
|
|
Standards
Undergraduate
|
|
Investigations and Activities
1. Compare selected nucleic acid sequences for Mouse and Human globin molecules
2. Create a Consensus Key
* - single, fully conserved residue
- no consensus
|
|
Resources
Pawliuk, R. et al, “Correction of sickle cell disease in transgenic Mouse Models by Gene Therapy”, Science 294:2368-2371, Dec 15, 2001
Sickle Cell Anemia. BluePrint for Health Visited 10/10/00. http://blueprint.bluecrossmn.com/topic/sickle.
Excellent overview of Microarray chips: http://www.gene-chips.com/
Students will usually obtain additional references or resources
to help answer or explore their questions. |
|
|
Special Data Items
Sequence alignment
Mouse b-globin:
1 gttgtgttga cttgcaactt cagaaacaga catcatggtg cacctgactg atgctgagaa
61 gtctgctgtc tcttgcctgt gggcaaaggt gaaccccgat gaagttggtg gtgaggccct
121 gggcaggctg ctggttgtct acccttggac ccagcggtac tttgatagct ttggagacct
181 atcctctgcc tctgctatca tgggtaatcc caaggtgaag gcccatggca aaaaggtgat
241 aactgccttt aacgagggcc tgaaaaacct ggacaacctc aagggcacct ttgccagcct
301 cagtgagctc cactgtgaca agctgcatgt ggatcctgag aacttcaggc tcctgggcaa
361 tgcgatcgtg attgtgctgg gccaccacct gggcaaggat ttcacccctg ctgcacaggc
421 tgccttccag aaggtggtgg ctggagtggc cactgccctg gctcacaagt accactaagc
481 cccttttctg ctattgtcta tgcacaaagg ttatatgtcc cctagagaaa aactgtcaag
541 tgtggggaaa tgatgaagac ctttgggcat ctagctttta tctaataaat gatatttact
601 gtcatctcaa aaaaaaaaaa aaaaaaaaaa aa
human sickle b-globin:
1 acatttgctt ctgacatagt tgtgttgact cacaacccca gaaacagaca tcatggtgca
61 cctgactgat gctgagaagg ctgctgtctc tggcctgtgg ggaaaggtga acgccgatga
121 agttggtggt gaggccctgg gcaggctgct ggttgtctac ccttggaccc agcggtactt
181 tgatagcttt ggagacctat cctctgcctc tgctatcatg ggtaatgcca aagtgaaggc
241 ccatggcaag aaagtgataa ctgcctttaa cgatggcctg aatcacttgg acagcctcaa
301 gggcaccttt gccagcctca gtgagctcca ctgtgacaag ctgcatgtgg atcctgagaa
361 cttcaggctc ctgggcaata tgatcgagat tgtgctgggc caccacctgg gcaaggattt
421 cacccccgct gcacaggctg ccttccagaa ggtggtggct ggagtggctg ctgccctggc
481 tcacaagtac cactaacgcc ccttttctgc tattgtctat gcacaaaggt tatatgtccc
541 ctagagaaaa actgtcaatt gtggggaaat gatgaagacc tttgggcatc tagcttttat
601 ctaataaatg atatt
|
|
Student Products
1. sequence of both murine and human sickle b-globin genes
2. Determine percent homology
3. Redesign the probe to include the 387th base pair
|
|
Assessment and Evaluation Plan
1. sequence of murine and human sickle b-globin genes (10% each)
2. percent homology (20%)
3. probe redesign to include the sickle mutation (60%).
|
|
Implementation
|
Course name:
|
Introduction to Bioinformatics
|
|
Likely sequence in
syllabus:
|
Assignment given after covering nucleotide databases, homology, and microarrays
|
|
Time during term:
|
Week 10
|
|
Duration:
|
one week
|
|
Setting:
|
|
|
Students in course:
|
Senior level elective for biology/pre-med students
|
|
Collaborative elements:
|
|
|
Additional notes:
|
|
|
|
|
Credits
|