MOLECULAR DESIGN AND CLONING OF CONTROL SHRNA FOR THE EXPERIMENTS SILENCING HUMAN MACROPHAGE ELASTASE
Abstract
Changes in the expression of matrix metalloproteinase 12 (MMP12/MMP12) are essential for the
pathogenesis of several diseases. The induction of MMP12 occurs in burns and keloids. In psoriasis and
herpetiformis Duhring dermatitis, the expression of MMP12 precedes a remodeling of dermal microcapillaries.
Moreover, the elevated expression of MMP12 is one of the well-known risk factors for melanoma. The aim of this
study was to create an expression vector producing control shRNA (small hairpin RNA) for the experiments on
human MMP12 silencing. To compare the alternative variants of predesigned control shRNA, we used free
electronic resources "OligoCalc", "blastn suite", and "Palindromic Sequences Finder". The double-stranded DNA
encoding the desired shRNA sequence was obtained by annealing two single-stranded oligonucleotides. After
annealing, we cloned the obtained double-stranded DNA between EcoRI and BamH1 binding sites of the
expression vector pGPV-17019250 using the respective endonucleases and T4 DNA ligase. Using a computer
analysis, we designed the control shRNA, a proposed negative control for silencing human MMP12. Then, we
cloned the DNA sequence encoding the desired shRNA in the expression vector pGPV-17019250 to generate the
new vector pGPV-17019250-MMP12c. The obtained data suggest that the vector pGPV-17019250-MMP12c can
express the control MMP12 shRNA and serve as a negative control in the experiments designated to silence
MMP12 in human cells. In this paper, we present the new vector pGPV-17019250-MMP12c encoding a control
MMP12 shRNA suitable for its expression in human cells. The named shRNA can be used as a negative control
for the experiments on MMP12.
References
2. Stawski L, Haines P, Fine A, Rudnicka L, Trojanowska M. MMP-12 deficiency attenuates angiotensin II-induced vascular injury, M2 macrophage accumulation, and skin and heart fibrosis // PLoS One. 2014. V. 9. № 10. e109763.
3. Zhang Z, Zhu S, Yang Y, Ma X, Guo S. Matrix metalloproteinase-12 expression is increased in cutaneous melanoma and associated with tumor aggressiveness // Tumour Biol. 2015. V. 36. № 11. P. 8593 – 8600.
4. Mogulevtseva, J.A., Mezentsev, A., Bruskin S.A., The Role of Matrix Metalloproteinases in the Pathogenesis of Psoriasis, in A Closer Look at Metalloproteinases. Hauppauge, NY. Nova science publishers, 2019.
5. Koolwijk P, Sidenius N, Peters E, Sier C.F., Hanemaaijer R, Blasi F., van Hinsbergh V.W. Proteolysis of the urokinase-type plasminogen activator receptor by metalloproteinase-12: implication for angiogenesis in fibrin matrices // Blood. 2001. V. 97. № 10. P. 3123 – 3131.
6. Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose // Proc. Natl. Acad. Sci. USA. 1979. V. 76. № 2. P. 615 – 619.
7. Bertani G. Lysogeny at mid-twentieth century: P1, P2, and other experimental systems // J. Bacteriol. 2004. V. 186. № 3. P. 595 – 600.
8. Hammond M. P., Birney E. Genome information resources – developments at Ensembl // Trends Genet. 2004. V. 20. № 6. P. 268 – 272.
9. Bondar' V., Mezencev A.V. Klonirovanie posledovatel'nosti shRNK, specifichnoj k jelastaze makrofagov, v jekspressionnyj vektor pGPV-17019250 // Evrazijskij Sojuz Uchenyh. Serija: medicinskie, biologicheskie i himicheskie nauki. 2021. № 7. S. 29–36.
10. Online Application to design scrambled sequence [Electronic resource]. URL: https://www.genscript.com/ssl-bin/app/scramble (Accessed at 18.01.2022).
11. Scrambled siRNA [Electronic resource]. URL: http://www.invivogen.com/sirnawizard/scrambled.php (Accessed at 18.01.2022).
12. Oligo Calc [Electronic resource]. URL: http://biotools.nubic.northwestern.edu/OligoCalc.html (Accessed at 18.01.2022).
13. blastn suite [Electronic resource]. URL: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE =BlastSearch (Accessed at 18.01.2022).
14. Palindromic Sequences Finder [Electronic resource]. URL: https://www.novoprolabs.com/tools/dna-palindrome (Accessed at 18.01.2022).
15. Design hairpin insert [Electronic resource]. URL: https://www.invivogen.com/sirnawizard/construct.php (Accessed at 18.01.2022).
16. Kristen A.V., Ajroud-Driss S., Conceição I., Gorevic P., Kyriakides T., Obici L. Patisiran, an RNAi therapeutic for the treatment of hereditary transthyretin-mediated amyloidosis. // Neurodegener Dis. Manag. 2019. V. 9. № 1. P. 5-23.
17. Geall A.J., Verma A., Otten G.R., Shaw C.A., Hekele A., Banerjee K., Nonviral delivery of selfamplifying RNA vaccines // Proc Natl Acad Sci U S A. 2012. V. 109. № 36. P. 14604 – 14609.
18. Bogenhagen D.F., Prinz K.G. The action of DNA ligase at abasic sites in DNA // J. Biol Chem. 1998. V. 273. № 14. P. 7888-7893.
19. Green, M.R., Sambrook J. Molecular Cloning: A Laboratory Manual: 4th ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2012. P. 235-239
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