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- 1. Zinc Finger Nuclease (ZFNs) (Generation of mastitis resistance in cows by targeting human lysozyme gene to β-casein locus using zinc-finger nucleases) Ng Ming Hui (0335713) Lee Kang Qi (0332311) Chong Hui Peng (0337206) Chu Sin Nin (0336511) Steven Noble (0335772)
- 2. WHAT IS ZINC FINGER NUCLEASE (ZFNs) ? Artificial proteins which are comprised of so-called zinc-finger peptides, which recognize a DNA sequence, and a nuclease (Fok1), which cuts the DNA. zinc-finger nucleases act in pairs on two sites close to each other in order to cut (enables the catalytic action of the Fok1 enzymes) By designing two zinc finger motifs recognizing either side of 5–6 bp spacer sequences at a target region, FokI nuclease combined with zinc finger can introduce DSBs within a target region (Urnov et al., 2010).
- 3. Repair by ligate the break end without the template HOW ZFNs WORK Repair using donor DNA as a template HOMOLOGOUS RECOMBINATION NONHOMOLOGOUS END JOINING
- 4. CHALLENGEADVANTAGES 1, Rapid disruption of, or integration into, any genomic loci 2. Mutations made are permanent and heritable 3.Works in a variety of mammalian somatic cell types 4. Edits induced through a single transfection experiment 5. Knockout or knock-in cell lines in as little as two months 1. Off target cleavage might occur 2. Immunological response toward the foreign protein inserted. 3. Hard or difficulties in construction of zinc finger domain 4. Limited target site selection - to knock in specific mutation. 5. Low efficiency WHY ZFNs AND WHY NOT
- 5. CURRENT DEVELOPMENT OF ZFNs 01 Evolution od DNA binding domain. Increase the ZFN fingers to improve specificity. Addition of the proteasomal inhibitor MG132. 02 Directed evolution of the FokI cleavage domain (FCD). To reduce off-target site cleavages and reduce toxicity. 04 ZFN delivery. Allowed the endocytosis and internalization of ZFNs by conjugating transferrin and ZFNs 03 Modification of cell culture method.
- 6. OVERVIEW Mastitis is a condition which causes a woman's (in this case female cow) breast tissue to become painful and inflamed. OBJECTIVE(S) - To design an efficient and reproducible gene- targeting system in bovine fetal fibroblasts (BFFs). - Producing transgenic cows expressing human lysozyme. - determine an effective procedure to prepare human lysozyme transgenic cells with ZFNs for SCNT.
- 7. ZFNs targeting intron 2 of the bovine CSN2 gene and plasmid pEGFP-I-hLYZ were constructed CONSTRUCTION OF ZFN AND DONOR DNA CELL CULTURE AND SELECTION MATERIAL AND METHOD Drug-resistant colonies were screened. Then run Western Blotting. DNA AND PROTEIN ANALYSIS Milk was collected and centrifuged. The collected infranatant was tested immediately MILK COLLECTION AND BACTERIAL CHALLENGE 03 01 05 Isolation of cell and electroporation with the plasmid then mixed with fresh medium. Do PCR screening after 9 days. SCNT were performed. Southern blot were done using the calf ear biopsies. NUCLEAR TRANSFER AND SOUTHERN BLOT ANALYSIS 02 04
- 8. ANALYSIS OF CLONED COW screens for each locus revealed patterns consistent with targeting . both 5′ (external) and hLYZ coding sequence (internal) probes hybridized to restrict fragments of the correct size. 1 JUNCTION PCR 2 SOUTHERN BLOT ANALYSIS Of viable bacterial cultures to test the transgenic cows’ ability to resist bacteria infection. 3 INTRA-MAMMARY INFUSION
- 9. DISCUSSION Limitation of Direct Injection Difficulties in raising of recombinant protein production level. Gene targeting :ZFN Donor Cells for NT: gene targeted cell lines Solving Method Nuclear transfer (NT), Cloning of gene- transfected somatic cells Production of precisely engineered transgenic cow Purpose
- 10. OUTCOME Inserted Successfully When these cows lactated, the milk contained both bovine and human lysozymes that had stronger antibacterial activity than non-transgenic cows. Hencevr enhance resistance to disease and improving the health and welfare of livestock RESULTS 5 Transgenic cows were produce Born July 2010 Of lysozyme typically 0.05–0.22 μg/ml LYSOZYME ACTIVITY 1/10 COMPARE TO HUMAN FYI
- 11. References (1) Genetherapynet.com. (2011). Zinc Finger Nuclease as Gene Editing Tool. [online] Available at: http://www.genetherapynet.com/gene-editing-tools/zinc-finger-nuclease.html [Accessed 5 Oct. 2019]. Gupta, R.M. and Musunuru, K. (2014). Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9. Journal of Clinical Investigation, [online] 124(10), pp.4154–4161. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4191047/. Ito Y, Yamada H, Nakamura M, Yoshikawa A, Ueda T& Imoto T. 1993. The primary structures and properties of non-stomach lysozymes of sheep and cow, and implication for functional divergence of lysozyme. Eur. J. Biochem. FEBS 213, 649–658. Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, Gregory PD, Pabo CO, Rebar EJ (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25(7):778–785. Mushtaq, Muntazir & Bhat, Javaid & Mir, Zahoor & Sakina, Aafreen & Ali, Sajad & Singh, Anil & Tyagi, Anshika & Salgotra, Romesh & Dar, Ahmad & Bhat, Rohini. (2018). CRISPR/Cas approach: A new way of looking at plant-abiotic interactions. Journal of Plant Physiology. 224. 10.1016/j.jplph.2018.04.001.
- 12. References (2) NHS Choices (2019). Overview - Mastitis. [online] NHS. Available at: https://www.nhs.uk/conditions/mastitis/ [Accessed 9 Oct. 2019]. Pavletich, N. P., and Pabo, C. O. (1991). Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science 252, 809–817. doi: 10.1126/science.2028256 Piccinini R, Binda E, Belotti M, Casirani G& Zecconi A. 2005. Comparison of blood and milk non-specific immune parameters in heifers after calving in relation to udder health. Vet. Res. 36, 747–757. Ramakrishna S, Kim YH, Kim H (2013) Stability of zinc finger nuclease protein is enhanced by the proteasome inhibitor MG132. PLoS One 8(1):e54282. Szczepek M, Brondani V, Buchel J, Serrano L, Segal DJ, Cathomen T (2007) Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol 25(7):786–793. Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., and Gregory, P. D. (2010). Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646. doi: 10.1038/nrg2842
- 13. Does anyone have any questions? minghui991127@gmail.com +60 17-669 1695 THANKS
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Editor's Notes
- 1.These artificial proteins are comprised of so-called zinc-finger peptides, which recognize a DNA sequence, and a nuclease (Fok1), which cuts the DNA. 2. consists of two beta sheets and an alpha helix, with one or more coordinated zinc ions at their core to confer rigidity to finger (Pavletich and Pabo, 1991). 3. a zinc-finger peptide unit recognizes 3 bp of DNA 4. zinc-finger nucleases act in pairs on two sites close to each other in order to cut (enables the catalytic action of the Fok1 enzymes) 5. By designing two zinc finger motifs recognizing either side of 5–6 bp spacer sequences at a target region, FokI nuclease combined with zinc finger can introduce DSBs within a target region (Urnov et al., 2010).
- A pair of three-finger ZFNs is shown at the top in association with a target gene (open box). If a homologous donor DNA is provided (solid box, left), repair can proceed by homologous recombination using the donor as template. The amount of donor sequence ultimately incorporated will typically decline with distance from the original break, as illustrated by the shading. Alternatively, the break can be repaired by nonhomologous end joining, leading to mutations at the cleavage site. These may be deletions, insertions, and base substitutions, usually quite localized, but sometimes extending away from the break.
- Rapid disruption of, or integration into, any genomic loci Mutations made are permanent and heritable Works in a variety of mammalian somatic cell types Edits induced through a single transfection experiment Knockout or knock-in cell lines in as little as two months Single or biallelic edits occur in 1–20% of clone population No antibiotic selection required for screening Off target cleavage might occur, if the zinc finger domains are not specific enough for their target site, which might result in cell death or apoptosis. Immunological response toward the foreign protein inserted. Hard or difficulties in construction of zinc finger domain that bind with high affinity toward the desired sequence, it is laborious, time consuming, and expensive. Limited target site selection - to knock in specific mutation. Low efficiency
- 1. ZFNs were designed to have three fingers in the binding domain, a structure that would individually recognize a 9-bp target site. Although, 3-finger ZFN was efficient enough to specify a unique recognition sites, ZFN that contains four, five, or six ZF modules is also made to further improve the specificity. 2. the formation of ZFN homodimers, which can activate FokI cleavage domain (FCD) and result in off-target site cleavages. To overcome this negative side effect, a new system was developed in such that FCD becomes active only when in the heterodimeric form. Such obligatory heterodimeric ZFNs showed a higher specificity with significantly reduced off-target site cleavages and reduced toxicity than those containing the wild-type FCD. 3. the ZFN protein is ubiquitinated and degraded by the proteasome, with a half-life of about 2 h. Addition of the proteasomal inhibitor MG132 extended the half-life up to 5 h and increased ZFN protein levels in the cells in a dose-dependent manner, leading to an enhanced ZFN activity. 4. By conjugating transferrin and ZFNs, which bind to ubiquitous plasma membrane transferring receptor, they successfully allowed the endocytosis and internalization of ZFNs. They showed that this method was efficient enough to create site-specific cleavage at the targeted locus and gene correction with better temporal control over level of ZFN activity in shorter period.
- Mastitis is a condition which causes a woman's (in this case female cow) breast tissue to become painful and inflamed. Mastitis costs the dairy industry billions of dollars annually and is the most consequential disease of dairy cattle. Transgenic cows secreting an antimicrobial peptide demonstrated resistance to mastitis. to design an efficient and reproducible gene-targeting system in bovine fetal fibroblasts (BFFs) by inserting the exogenous human lysozyme (hLYZ) gene into the β-casein locus with ZFNs and subsequently using the targeted cell clones as donor cells for somatic cell nuclear transfer (SCNT). transgenic cows expressing human lysozyme in their milk are resistant to certain Gram-positive bacteria (e.g. Staphylococcus aureus) and Gram-negative bacteria infection. determine an effective procedure to prepare human lysozyme transgenic cells with ZFNs for SCNT that consists of evaluating the expression of transgenic constructs in bovine mammary epithelial cells (BMECs) in vitro and identifying competent transgenic fibroblast cell clones.
- 1, ZFNs targeting intron 2 of the bovine β-casein ( CSN2) gene was constructed. The ZFN cut site (5′-AGTAT-3′) in the centre of the ZFN-binding site. The plasmid pEGFP-I-hLYZ was constructed through a series of process. 2. Primary fetal bovine fibroblasts were isolated from 35-day-old fetuses. cells were placed in a gap cuvette with the targeting vector (pCSN2-hLYZ-Neo-GFP) and the ZFNs-encoding plasmids, and electroporated. Electroporated cells were mixed with fresh cell culture medium and plated. Cell clones were then collected for PCR screening after 9 days 3.Drug-resistant colonies were trypsinized and screened for targeting events by PCR. Mammary epithelial cells expressing green fluorescent protein were lysed and separated by SDS-PAGE. Used antibody to detect GFP, hLYZ, and beta casein. 4.SCNT procedures were performed. Pregnancy was detected by rectal palpation/ultrasonography at 90 days of gestation. Southern blot were done using the calf ear biopsies. 5. Milk was collected and centrifuged. The collected infranatant was tested immediately. Before bacterial mammary gland challenge were done, The health of the animals was assessed. Then the bacterial challenge was executed. (to test the activities of lysozyme) We used the single-cell-derived clones that were all heterozygous for the human lysozyme gene knock-in at the β-casein locus for NT and finally obtained gene-targeted calves. Gene-targeted BFFs were used in somatic cell nuclear transfer.
- Junction PCR screens for each locus revealed patterns consistent with targeting Southern blot analyses, both 5′ (external) and hLYZ coding sequence (internal) probes hybridized to restrict fragments of the correct size intra-mammary infusion of viable bacterial cultures to test The transgenic cows’ ability to resist infection caused by Sta. aureus, E. coli or Str. agalactiae Pictures: (a) PCR analysis carried out on chromosomal DNA of non-transgenic cow (lane 1) and of five gene-targeted calves lived for more than one month (lanes 2–6). 5′ junction PCR should yield 1281 bp PCR products using primers P1 and P2 (upper), 3′ junction PCR should yield 1337 bp PCR products using primers P3 and P4 (lower). The positions and sizes of specific PCR products are indicated at the right. (b) Southern blot analysis of the CSN2 knock-in calves. Lane 1 contains normal cow DNA digested with BglII as a negative control. Lanes 2–6 are BglII-digested genomic DNA from five gene-targeted calves. Using the external probe 5′ of the genome homology region, the CSN2 knock-in calves showed two bands: a 7.5 kb band from the endogenous CSN2 allele and a 5.2 kb band characteristic of the insertion (upper). The 5.2 kb band was also detected with an hLYZ probe (lower). Table: Of the mammary glands infused, 19 of 20 glands became infected in non-transgenic animals compared with 0 of 20 glands in transgenic animals
- To enhance mastitis resistance, antibacterial protein is produced by direct injection of DNA into mammary gland of transgenic animal. However it comes with limitation which is Raising of recombinant protein production level in mammary gland is difficult because: The injection of exogenous DNA into animal genome is integrated randomly Integration rate in the genomic DNA is really low Solving method: Nuclear Transfer (NT). In ZFNs, bovine fetal fibroblasts (BFFs) is used as donor cells in gene targeted cell lines. By cloning gene-transfected somatic cells, transgenic domestic animals have been produced.
- We have successfully inserted the human lysozyme gene into the β-casein gene locus of cows. Bovine milk typically contains only 0.05–0.22 μg ml−1 of lysozyme (Piccinini et al., 2005). In addition, its activity is 1/10 of lysozyme from human breast milk (Ito et al., 1993). When these cows lactated, the milk contained both bovine and human lysozymes that had stronger antibacterial activity than non-transgenic cows. To enhance resistance to disease and improving the health and welfare of livestock and establish effective procedure to prepare transgenic donor cells by gene targeting for SCNT.