Ann Hillam and Dr. Laura C. Bridgewater, Zoology

The Col11a2 gene encodes the production of type XI collagen which is fundamental for normal skeletal development. As with many genes, comparatively little is known of the transcriptional activation and enhancement mechanisms of the Col11a2 gene. Such knowledge will help advance gene therapy techniques to minimize and eventually eradicate the negative physical defects effectuated by a mutated Col11a2 gene.

Enhancer elements are DNA sequences responsible for activating a particular gene=s transcription. Discovering how many enhancer elements are involved in initiating transcription of a specific gene and knowing where these enhancers are located are fundamental to understanding that gene. Currently three enhancers have been located in conjunction with this gene. For my ORCA research, I searched a small region of the Col11a2 gene=s first intron sequence for additional enhancer elements. The region I tested was approximately 230bp (DNA base pairs) long, and the three basic steps of this experiment involved the following:

1. Excise two fragments, approximately 115bp each, individually from the intron sequence.
2. Clone the two altered intron sequences into the pCXI wt 2/3 vector (a circular bacterial DNA also known as a plasmid or plasmid vector) preceding the â-galactosidase gene using the SalI, BglII, and PstI restriction enzyme sites and sticky end ligation.
3. Test the reconstructed vectors in tissue cultureBspecifically, in rat chondrosarcoma (RCS) cells by transfection using LipofectAMINEBfor â-galactosidase gene transcription activity.

Polymerase chain reaction (PCR) was used to excise the115bp fragments by replicating only the desired sequence(s) from the intron. I constructed primers similar in GC content that contained either SalI or SalI and BglII in the forward primer and PstI or BglII as part of the reverse primer sequence. The first 115bp of the intron was not replicated during one PCR reaction to create a product of approximately 591bp named intdel.7 or insert 1. To delete the next 115bp of the intron, two PCR products were made and named intdel.8 as a whole or insert 2.1 and insert 2.2 individually. The first PCR product was around 132bp (insert 2.2) and contained the beginning of the intron sequence, then a segment of about 487bp (insert 2.1) following the deletion was replicated. Of the three final PCR products, insert 1 had the SalI and PstI restriction enzyme sites included at its 5′ and 3′ ends respectively, insert 2.1 contained SalI and BglII at its 5′ end and PstI at its 3′ end, and insert 2.2 included a SalI 5′ restriction enzyme sequence and BglII site at the 3′ end. These specific enzymes and directionality were used so that the intron inserts could be properly placed into the pCXI wt 2/3 vector. Each PCR product was run on an agarose gel to check for correct replication and amplification. PCR was redone on those samples that did not work until satisfactory yields and the right amount of base pairs had been replicated, which would suggest correct amplification of the intron sequence. Each individual PCR product was then excised from the gel and purified for use in the second step of this experiment.

For the second part of this research project, I ligated intdel.7 and intdel.8 individually into the pCXI wt 2/3 vector, then used transformation into Esherichia coli bacteria to clone the vector. First, the circular vector was cut with SalI and PstI restriction enzymes, then inserts 1 and 2.1 were added to the vector in different reactions using sticky end ligation. To clone insert 2.2 into the vector containing insert 2.1, the vector had to be re-cut, but with BglII and PstI. Insert 2.2 was then ligated into this vector using sticky end ligation. To test that the inserts had correctly ligated to the vector, a small sample of each construct was re-cut with the necessary enzymes, then run on an agarose gel. As long as the ligation had been successful, I saw one band of approximately 8kb representing the vector and smaller bands of either 591bp, 487bp, or 132bp depending on which inserts had been added. I had trouble cloning insert 2.2 into the vector so it was necessary to alter the ratio of insert to vector several times before it ligated properly. With this done, however, I was then able to clone each vector construct in E. coli bacteria, pick several colonies to test for proper transformation, then prepare the cloned vectors for use in cell transfection.

Rat chondrosarcoma (RCS) cells were used for the tissue culture assay to test the vector construct for activity of the â-galactosidase gene. I used both â-galactosidase and luciferase to measure âgalactosidase transcription, and LipofectAMINE to transfect the RCS cells with the vector constructs. The positive control used was the pCXI wt 2/3 vector containing the â-galactosidase gene, but no intron inserts, and the vector pNASSB was used as the negative control. Due to time constraints, I was only able to successfully transfect the RCS cells once, and was therefore unable to re-do the transfection to verify that my results were accurate. Another student in Dr. Bridgewater’s lab picked up where I let off and did transfection four separate times. The averages of their results showed that the average percent of â-galactosidase gene activation was 100% in the positive control, .8% in the negative control, 115.25% in intdel.7, and 55.25% in intdel.8. This data suggests that part or all of an enhancer element sequence is encoded within the region of 116-230bp of the intron because of a reduction in gene function by nearly 45% compared to normal. Because of this data, Dr. Bridgewater and those working with her are researching the exact location and sequence of this enhancer element.

The difficulties I faced in completing this research were due either to lack of experience or time. Technical errors on my part involved re-doing some aspects of the experiment one or multiple times, and because I was graduating in April, I did not have the time to re-test my results from tissue culture and transfection. However, conducting the research for this ORCA scholarship has been invaluable to me, not only in my undergraduate endeavors, but it has helped me qualify for a position as a laboratory technician within a human genetics laboratory at Harvard Medical School. I have enjoyed using this experience to increase my knowledge and awareness of laboratory research. I hope this program will continue to be a source of motivation and knowledge for many future students, and I’m grateful for the opportunity I had to participate in the ORCA scholarship program.