What is gene trapping? Technique used randomly disrupts genes throughout the genome. Insert a DNA element: reporter gene & selectable marker. Between two exons, both sites splicing (reporter and exon),’’fusion mRNA’’ transcription , reporter gene: treated like exon [2].Correct farm of the exon, reporter expressed in the similar pattern as the endogenous gene. Produced functional tag [3]. Used an objective, economic & high-throughput tool to dissect genetic influences on cell function The vector splice acceptor, splice donor sequences& antibiotic resistance gene . Introduction Genetic studies have highlighted the difference in the level of gene frequently and gene variety in tumour, where each clone different from another genetic feature context. In fact, cancer causes alteration in the gene fitness in each single clone due to genetic variation which may occurred during a series of fluctuation of clonal growth. However, cancer have the ability to evolve resistance to traditional therapies leads to serious problem in both primary and secondary tumour, for example, a wide range of responses were recorded in patients as a results of genetic alteration responsible for drug resistance. Further, studying the classical chemotherapy individually or in combination by using new techniques and method is quite important to design and discover anticancer drugs with molecular properties, integrating directly with the function of the genes [1] . Conclusions Although it is complicated process and time consuming, but still gene trapping techniques are one of the promising methods in the genetic analysis, applying randomly to investigate on unknown mutated gene, sequencing and identifying it, and then understand the molecular behaviours and study the phenotypic properties by utilizing other techniques such as microarray. Indeed, the application of automated methods will be evolving this techniques, increasing the efficiency and accuracy. Future work • Personalized disease diagnosis: depends on the bio-marks: proteins, enzymes .etc. which their levels may be changed during the genes mutation. • Targeting the gene by knockdown (oncogenes), while activate tumour suppresser genes such as:BRCA1and BRCA2, in breast cancer. • Screening and Consulting for family members of affected individual. Figure 1. The integration of the gene trap vector into the pre-mutant cell’s genome[2]. . Background and Aim : Cisplatin: widely used as chemotherapy in various type of cancer diseases. Details of the molecular mechanism responsible for cisplatin resistance are unclear. The goal of this research was to identify the expression of genes related to cisplatin resistance in (HEK293)cell line, which can used in future to investigate the resistance phenotype. Method and Procedure : Figure 2. Eight mutated HEK293 cell lines that show resistance to the cytotoxic effects of cisplatin. Each was subject to RACE-PCR to identify the gene mutation responsible for survival. Figure 3. Scheme shows method and procedure of the research: HEK293 cells were mutated with the pGTVI3 gene trap vector. Productive mutagenic insertions in the HEK293 genome treated with cisplatin, after did cisplatin concentration assessment, expanding and splitting the ‘gene trap library’ into ‘control’ and ‘Cisplatin’ sets of flasks. 1-Cisplatin preparation: 2- Cell line culture, Media, Trypsin, Feeding, Freezing, and recovery. 3- Insertion of pG TIV3. 4 - Concentration Assessment: 5 -HEK293 Gene Trapping Library. 6- Immunofluorescence Slides. 7- Mutation Identified: 7. A) RNA Extraction: using RNA Kit (BioLine, Life Science ® Company) 7. B) cDNA 7 .C) RACE1 and RACE2 7 .E) Purification 7. F) DNA ligation 7.G) Transformation and Cloning of Plasmid 7.H) Digest with EcoRI-insert 7.I) sequencing RACE 2 products in plasmids were sent for sequencing by Source Bioscience (Nottingham, UK). The results were identified by using the UCSC BLAT tool. References [1] Geddes JR, Miklowitz DJ(2013) Lancet 381:1672-82 [2] Gow M, Mirembe D, Longwe Z, Pickard BS (2013) J Cell Mol Med. 17:657-63. [3] Psychiatric GWAS Consortium Bipolar Disorder Working Group (2011) Nat Genet. 43:977-83. [4] USCU genome website : [5] Davies S, Lopez Sanchez M, Narsai R, Shearwood A, Razif M, Small I, Whelan J, Rackham O, Filipovska A.(2012).MRPS27 is pentatricopeptid repeat protein required for the translation of mitochondrially encoded protiens . FEBS Letter 586, 3555-3561. [6] Akimaru K, Kuo MT, Furuta K, Suzuki M, Noyori R, Ishikawa T. (1996). Cytotechnology. , 19(3):221-7. [7] Spugnini, E.,Citro, G., and Fais, S. (2010). Proton pump inhibitors as anti-vascular-ATPase drugs: anovel anticancer strategy. Journal of Experiment &Clinical Cancer Research, 29-44. [8] de Larrea C, Navarro A, Tejero R, Tovar N, Díaz T, Cibeira Ferrer G, Rovira M, Rozman M, Monzó M, Bladé J(2012). Impact of MiRSNPs on surival and progression in patientswith multiple myeloma undergoing autologous stemm cell transplantation. Clin Cancer, Jul 1; 18(13):3697-3704 [9] Maddirala Y., Tobwala S., Ercal N. ( 2015 ). N- acetylcysteineamide protects against manganese-induced toxicity in SHSY5Y cell line. Brain Res., 22;1608:157-66. Figure4. Scheme Shows the future clinical implications . Results / Four genes were identified : [MRP27S, MVB15B, FAM179, and NACA] . Acknowledgments: My sincere thanks goes to Dr Elizabeth Ellis, I prayer to her to rest in peace. I would to express my sincere gratitude to my supervisor Dr Benjamin Pickard for unlimited support and encouragement during my MSc study and related research MRPS27 Ch:5 (q13,2) Related to (PPR: Pentatricopeptide repeat Family). (1st member was LRPPRC) [4], [5], [6]. Mitochondrial translation protein, known to associate with Death Associated Protein 3 (DAP3): Apoptosis role? Nuclear gene encoding mitochondrial protein, mRNA.(28S ribosomal protein S27, mitochondrial). mitochondrial translation Source, mitochondrial translational elongation Source, mitochondrial translational initiation Source , mitochondrial translational termination Source, organelle organization. MVB12B. multivesicular bodies) transcript variant 1, mRNA [7]. In humans, the structurally similar subtypes MVB12A and MVB12B are subunits of ESCRT-I. (Endosomal Sorting Complex Required for Transport pathway). MVB12B is a ubiquitinated protein, The ubiquitinations sites in MVB12B are Lys264 and Lys290. Depletion and overexpression of this and related protein (MVB12A) inhibit HIV-1 infectivity and induce unusual viral assembly defects, indicating a role for MVB12 subunits in regulating ESCRT-mediated virus budding. FAM179B [8]. Homo sapiens family with sequence similarity 179, member B (FAM179B), mRNA. • This gene at miRNA. • Interact with single nucleotide polymorphism (SNP) molecules at the active site to form a complex (miR SNPs) with DNA target site, which compete the reaction of cisplatin. This is an example about DNA repair by Nucleotide Excision Repair (de Larrea, et al., 2012) . NACA [ch12(q13,3)] [9]. Nascent polypeptide-associated complex alpha subunit (NACA): stops incorrect targeting of proteins, also role in apoptosis?? • Homo sapiens nascent polypeptide-associated complex alpha subunit (NACA), transcript variant 1, mRNA. • Connected with basic transcription factor 3(BTF3), forming nascent polypeptide- associated complex (NAC). • The signal recognition particle (SRP) can be blocked when NAC binds with a nascent protein as they are from the same ribosomal precursor, coding to the endoplasmic reticulum.
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What is gene trapping?Technique used randomly disrupts genes throughout the genome. Insert a DNA element: reporter gene & selectable marker. Between two exons, both sites splicing (reporter
and exon),’’fusion mRNA’’ transcription , reporter gene: treated like exon [2].Correct farm of the exon, reporter expressed in the similar pattern as the endogenous gene.Produced functional tag [3]. Used an objective, economic & high-throughput tool to dissect genetic influences on cell function The vector splice acceptor, splice donorsequences& antibiotic resistance gene .
IntroductionGenetic studies have highlighted the difference in
the level of gene frequently and gene variety in tumour, where each clone different from another genetic feature context. In fact, cancer causes alteration in the gene fitness in each single clone due to genetic variation which may occurred during a series of fluctuation of clonal growth. However, cancer have the ability to evolve resistance to traditional therapies leads to serious problem in both primary and secondary tumour, for example, a wide range of responses were recorded in patients as a results of genetic alteration responsible for drug resistance. Further, studying the classical chemotherapy individually or in combination by using new techniques and method is quite important to design and discover anticancer drugs with molecular properties, integrating directly with the function of the genes [1] .
ConclusionsAlthough it is complicated
process and time consuming, but still gene trapping techniques are one of the promising methods in the genetic analysis, applying randomly to investigate on unknown mutated gene, sequencing and identifying it, and then understand the molecular behaviours and study the phenotypic properties by utilizing other techniques such as microarray. Indeed, the application of automated methods will be evolving this techniques, increasing the efficiency and accuracy.
Future work• Personalized disease
diagnosis: depends on the bio-marks: proteins, enzymes .etc. which their levels may be changed during the genes mutation.
• Targeting the gene by knockdown (oncogenes), while activate tumour suppresser genes such as:BRCA1and BRCA2, in breast cancer.
• Screening and Consulting for family members of affected individual.
Figure 1. The integration of the gene trap vector into the pre-mutant cell’s genome[2].
.
Background and Aim : Cisplatin: widely used as chemotherapy in various type of cancer diseases. Details of the molecular mechanism responsible for cisplatin resistance are unclear.
The goal of this research was to identify the expression of genes related to cisplatin resistance in (HEK293)cell line, which can used in future to investigate the resistance phenotype.
Method and Procedure :
Figure 2. Eight mutated HEK293
cell lines that show resistance to the cytotoxic effects of cisplatin. Each was subject to RACE-PCR to identify the gene mutation responsible for survival.
Figure 3. Scheme shows method and procedure of the research: HEK293 cells were mutated with the pGTVI3 gene trap vector. Productive mutagenic insertions in the HEK293 genome treated with cisplatin, after did cisplatin concentration assessment, expanding and splitting the ‘gene trap library’ into ‘control’ and ‘Cisplatin’ sets of flasks.1-Cisplatin preparation: 2- Cell line culture, Media, Trypsin, Feeding, Freezing, and recovery.3- Insertion of pG TIV3.4 - Concentration Assessment:5 -HEK293 Gene Trapping Library.6- Immunofluorescence Slides.7- Mutation Identified:7. A) RNA Extraction: using RNA Kit (BioLine, Life Science ® Company) 7. B) cDNA7 .C) RACE1 and RACE2 7 .E) Purification7. F) DNA ligation 7.G) Transformation and Cloning of Plasmid 7.H) Digest with EcoRI-insert7.I) sequencingRACE 2 products in plasmids were sent for sequencing by Source Bioscience (Nottingham, UK). The results were identified by using the UCSC BLAT tool.
References[1] Geddes JR, Miklowitz DJ(2013) Lancet 381:1672-82[2] Gow M, Mirembe D, Longwe Z, Pickard BS (2013) J Cell Mol Med. 17:657-63.[3] Psychiatric GWAS Consortium Bipolar Disorder Working Group (2011) Nat Genet. 43:977-83.[4] USCU genome website :[5] Davies S, Lopez Sanchez M, Narsai R, Shearwood A, Razif M, Small I, Whelan J, Rackham O, FilipovskaA.(2012).MRPS27 is pentatricopeptid repeat protein required for the translation of mitochondrially encoded protiens . FEBS Letter 586, 3555-3561.[6] Akimaru K, Kuo MT, Furuta K, Suzuki M, Noyori R, Ishikawa T. (1996). Cytotechnology. , 19(3):221-7.[7] Spugnini, E.,Citro, G., and Fais, S. (2010). Proton pump inhibitors as anti-vascular- ATPase drugs: anovel anticancer strategy. Journal of Experiment &Clinical Cancer Research, 29-44.[8] de Larrea C, Navarro A, Tejero R, Tovar N, Díaz T, Cibeira Ferrer G, Rovira M, Rozman M, Monzó M, Bladé J(2012). Impact of MiRSNPs on surival and progression in patientswith multiple myeloma undergoing autologous stemm cell transplantation. Clin Cancer, Jul 1; 18(13):3697-3704[9] Maddirala Y., Tobwala S., Ercal N. ( 2015 ). N-acetylcysteineamide protects against manganese-induced toxicity in SHSY5Y cell line. Brain Res., 22;1608:157-66.
Figure4.Scheme Shows the future clinical implications .
Results / Four genes were identified : [MRP27S, MVB15B, FAM179, and NACA] .
Acknowledgments:My sincere thanks goes to Dr Elizabeth Ellis, I prayer to her to rest in peace. I would to express my sincere gratitude to my supervisor Dr Benjamin Pickard for unlimited support and encouragement during my MSc study and related research
MRPS27 Ch:5 (q13,2)Related to (PPR: Pentatricopeptiderepeat Family). (1st member was LRPPRC) [4], [5], [6].
Mitochondrial translation protein, known to associate with Death Associated Protein 3 (DAP3): Apoptosis role?Nuclear gene encoding mitochondrial protein, mRNA.(28S ribosomal protein S27, mitochondrial).mitochondrial translation Source, mitochondrial translational elongation Source,mitochondrial translational initiation Source , mitochondrial translational termination Source, organelle organization.
MVB12B. multivesicular bodies) transcript variant 1, mRNA [7].
In humans, the structurally similar subtypes MVB12A and MVB12B are subunits of ESCRT-I.(Endosomal Sorting Complex Required for Transport pathway).MVB12B is a ubiquitinated protein, The ubiquitinations sites in MVB12B are Lys264 and Lys290. Depletion and overexpression of this and related protein (MVB12A) inhibit HIV-1 infectivity and induce unusual viral assembly defects, indicating a role for MVB12 subunits in regulating ESCRT-mediated virus budding.
FAM179B[8].
Homo sapiens family with sequence similarity 179, member B (FAM179B), mRNA.• This gene at miRNA.• Interact with single nucleotide polymorphism (SNP) molecules at the active site to form a
complex (miR SNPs) with DNA target site, which compete the reaction of cisplatin. This is an example about DNA repair by Nucleotide Excision Repair (de Larrea, et al., 2012) .
NACA[ch12(q13,3)][9].
Nascent polypeptide-associated complex alpha subunit (NACA): stops incorrect targeting of proteins, also role in apoptosis??• Homo sapiens nascent polypeptide-associated complex alpha subunit (NACA), transcript
variant 1, mRNA.• Connected with basic transcription factor 3(BTF3), forming nascent polypeptide-
associated complex (NAC).• The signal recognition particle (SRP) can be blocked when NAC binds with a nascent
protein as they are from the same ribosomal precursor, coding to the endoplasmic reticulum.
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