Production of CFTR Mutant Gene Model by Homologous Recombination System

Document Type : Original Article


1 Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3 Regenerative Medicine Group (REMED), Universal Scientific Education and Research Network (USERN), Tehran, Iran 4. Mom Fertility and Infertility Research and Innovation Center, Tehran, Iran

4 Mom Fertility and Infertility Research and Innovation Center, Tehran, Iran

5 Toxicological Research Center, Loghman-Hakim Hospital, Department of Clinical Toxicology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran


Objective: The most common mutation in cystic fibrosis (CF), (ΔF508-CFTR), results in impaired protein maturation,
folding and transportation to the surface of the cell. As a consequence of impaired protein maturation and/or transport from the extracellular matrix to the cell, different systems are influenced, including gastrointestinal system and glandular system, reproductive system and respiratory systems. CF models are essential tools to provide further knowledge of
CF pathophysiology. With this aim, we designed a transgenic CF model based on the homologous recombination (HR)
Materials and Methods: In this experimental study, a specifically designed construct containing the CFTR gene with F508del was cloned into a PTZ57R cloning vector and then the construct was transformed into the male pronucleus by microinjection after in vitro fertilization (IVF). Then the rates of blastocyst formation and embryonic development at
72 hours after IVF, were evaluated using the inverted microscope and the insertion of the construct was approved by
polymerase chain reaction (PCR) method.
Results: The CFTR gene was successfully cloned into the PTZ57R cloning vector and overall, from 22 injected cells, 5 blastocysts were observed after pronuclear injection of the CFTR gene construct. PCR verification of the blastocyst with CFTR-specific primers represented complete recombination of CFTR into the mouse genome.
Conclusion: For the first time we designed a unique genome construction that can be detected using a simple PCR method. The pronuclear injection was performed for the transformation of the genome construct into the male pronuclei using microinjection and the development of zygote to the blastocyst stage has been observed following transgenesis.


  1. Lopes-Pacheco M. CFTR modulators: the changing face of cystic fibrosis in the era of precision medicine. Front Pharmacol. 2020; 101662.
  2. Wang Y, Wrennall JA, Cai Z, Li H, Sheppard DN. Understanding how cystic fibrosis mutations disrupt CFTR function: from single molecules to animal models. Int J Biochem Cell Biol. 2014; 52: 47-57.
  3. McCarron A, Donnelley M, Parsons D. Airway disease phenotypes in animal models of cystic fibrosis. Respir Res. 2018; 19(1): 1-12.
  4. Hodson ME, Simmonds NJ, Warwick WJ, Tullis E, Castellani C, Assael B, et al. An international/multicentre report on patients with cystic fibrosis (CF) over the age of 40 years. J Cyst Fibros. 2008; 7(6): 537-542.
  5. Rosen BH, Chanson M, Gawenis LR, Liu J, Sofoluwe A, Zoso A, et al. Animal and model systems for studying cystic fibrosis. J Cyst Fibros. 2018; 17(2): S28-S34.
  6. Yu L, Batara J, Lu B. Application of genome editing technology to microrna research in mammalians. Kormann MSD, editor. Modern tools for genetic engineering. London: IntechOpen; 2016.
  7. Leavitt AD, Hamlett I. Homologous recombination in human embryonic stem cells: a tool for advancing cell therapy and understanding and treating human disease. Clin Transl Sci. 2011; 4(4): 298-305.
  8. Yu H, Wang X, Zhu L, He Z, Liu G, Xu X, et al. Establishment of a rapid and scalable gene expression system in livestock by sitespecific integration. Gene. 2013; 515(2): 367-371.
  9. Rogers CS, Hao Y, Rokhlina T, Samuel M, Stoltz DA, Li Y, et al. Production of CFTR-null and CFTR-ΔF508 heterozygous pigs by adeno-associated virus–mediated gene targeting and somatic cell nuclear transfer. J Clin Invest. 2008; 118(4): 1571-1577.
  10. Engelhardt JF, Yankaskas JR, Ernst SA, Yang Y, Marino CR, Boucher RC, et al. Submucosal glands are the predominant site of CFTR expression in the human bronchus. Nat Genet. 1992; 2(3): 240-248.
  11. Sun X, Sui H, Fisher JT, Yan Z, Liu X, Cho HJ, et al. Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis. J Clin Invest. 2010; 120(9): 3149-3160.
  12. Xu J, Livraghi-Butrico A, Hou X, Rajagopalan C, Zhang J, Song J, et al. Phenotypes of CF rabbits generated by CRISPR/Cas9- mediated disruption of the CFTR gene. JCI Insight. 2021; 6(1): e139813.
  13. Dreano E, Bacchetta M, Simonin J, Galmiche L, Usal C, Slimani L, et al. Characterization of two rat models of cystic fibrosis—KO and F508del CFTR—Generated by Crispr-Cas9. Animal Model Exp Med. 2019; 2(4): 297-311.
  14. Leenaars CH, De Vries RB, Heming A, Visser D, Holthaus D, Reijmer J, et al. Animal models for cystic fibrosis: a systematic search and mapping review of the literature–Part 1: genetic models. Lab Anim. 2020; 54(4): 330-340.
  15. National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals. 8th ed. Washington (DC): National Academies Press (US); 2011.
  16. Zeng F, Hao Z, Li P, Meng Y, Dong J, Lin Y. A restriction-free method for gene reconstitution using two single-primer PCRs in parallel to generate compatible cohesive ends. BMC Biotechnol. 2017; 17(1): 32.
  17. Vahdat-Lasemi M, Hosseini S, Jajarmi V, Kazemi B, Salehi M. Intraovarian injection of miR-224 as a marker of polycystic ovarian syndrome declines oocyte competency and embryo development. J Cell Physiol. 2019; 234(8): 13858-13866.
  18. Colledge WH, Abella BS, Southern KW, Ratcliff R, Jiang C, Cheng SH, et al. Generation and characterization of a delta F508 cystic fibrosis mouse model. Nat Genet. 1995; 10: 445-452. 19. Scholte BJ, Davidson DJ, Wilke M, De Jonge HR. Animal models of cystic fibrosis. J Cyst Fibros. 2004; 3 Suppl 2: 183-190.
  19. Dunham MA, Neumann AA, Fasching CL, Reddel RR. Telomere maintenance by recombination in human cells. Nat Genet. 2000; 26(4): 447-450.
  20. Jasin M, Rothstein R. Repair of strand breaks by homologous recombination. Cold Spring Harb Perspect Biol. 2013; 5(11): a012740.
  21. Symington LS, Rothstein R, Lisby M. Mechanisms and regulation of mitotic recombination in Saccharomyces cerevisiae. Genetics. 2014; 198(3): 795-835.
  22. Popova E, Krivokharchenko A, Ganten D, Bader M. Efficiency of transgenic rat production is independent of transgene-construct and overnight embryo culture. Theriogenology. 2004; 61(7-8): 1441-1453.
  23. Sosa MAG, De Gasperi R, Elder GA. Animal transgenesis: an overview. Brain Struct Funct. 2010; 214(2-3): 91-109.
  24. Meyer M, de Angelis MH, Wurst W, Kühn R. Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci USA. 2010; 107(34): 15022-15026.
  25. Yan BW, Zhao YF, Cao WG, Li N, Gou KM. Mechanism of random integration of foreign DNA in transgenic mice. Transgenic Res. 2013; 22(5): 983-992.
  26. Bouabe H, Okkenhaug K. Gene targeting in mice: a review. Methods Mol Biol. 2013; 1064: 315-336.
  27. Hall B, Limaye A, Kulkarni AB. Overview: generation of gene knockout mice. Curr Protoc Cell Biol. 2009; Chapter 19: Unit 19.12 19.12.1-17.
  28. Yaghobi Moghaddam MA, Dehghan Esmatabadi MJ. A review of artificial genetic constructs and their applications as positive controls. J Human Gen Genom. 2019; 3(1): e99853.