Chronic myeloid leukemia (CML) remains as a malignant clonal disorder featured by virtue of hyperproliferation of myeloid cells localized in peripheral blood, spleen as well as bone marrow . many cases of chronic myeloid leukemia (CML) are recognized amid blood tests did for another reason. patients with Chronic myeloid leukemia (CML) and BCR-ABL T315I mutation do not profit from treatment with presently accepted tyrosine kinase inhibitors . The aim of the Study to Clone the Gene of interest TROVE2 transcript variant 1 (which involve in CML) that extracted from KCL22 cell line into E.coli with pBluescript plasmid.
Chronic myeloid leukemia (CML) remains as a malignant clonal disorder featured by virtue of hyperproliferation of myeloid cells localized in peripheral blood, spleen as well as bone marrow . The diagnosis by molecular as well as cytogenetics methods relies on detecting Philadelphia chromosome, featuring reciprocal translocation t(9;22). The consequences of such translocation results in the BCR-ABL oncogene which in turn activates tyrosine kinase that produces abnormal protein activation ending up in deregulation of the hematopoietic stem cells that triggers myeloid cell hyper proliferation ending up to CML. On the other hand, each of the cancerous cell line is being characterized by virtue of intracellular or extracellular biomarkers .
The utilization of ‘autoimmune antigen’ for the treating Chronic Myeloid Leukemia acts as a probable alternative. Variety of autoantibodies from the serum autoimmune patients’, are available including those specific antibodies for nuclear antigens. Such antibodies are commonly termed as antinuclear antibodies (ANA). These ANA are indeed specific against autoantigen Ro also known as SS-A else anti-Ro antibodies. These serve as diagnostic marker for detecting the condition. Ro antigen contains two major proteins Ro52 (SSA1, TRIM21, else RNF81) along with Ro60 (TROVE2 else SSA2 ) . The expression levels of TROVE2 is being dramatically elevated in concurrence with the pathological stages representing upregulation with most of tumors. Whenever steps are being taken for knocking down such expression, it will be a great contribution .
The TROVE2 functions as a conserved RNA-binding protein. The essential function towards nuclear functioning comprises of interacting with misfolded smaller cellular RNA, whereas the role of cytoplasm encompasses binding with Y RNA. Such Ro60,Y RNA, together with other proteins produces ribonucleoprotein complex (RoRNP) inside human cells. The RoRNP takes part to control the quality of non-coding RNA that are misfolded along with decisive function for survival by virtue of avoiding autoimmunity. The gene indeed binds with noncoding Y RNA thereby turn out to be accessible to autoantibodies that take part in apoptosis. This TROVE2 forms a ring with central hole, offering larger RNA binding surface with access towards generously proportioned collection of possible RNAs, together with Y RNAs, variant 5S rRNAs, along with misfolded U2 sn RNA.
Ro60 is encoded by TROVE2 that remains as a ribonucleoprotein complex component with Ro52, E3 ubiquitin ligase, with the Pol-III transcriptional terminator La/SSB, along with the short noncoding Y RNAs (HY1-4). The Ro60 has domain that’s are specific towards RNA recognition for sake of Y RNAs nonetheless it binds auxiliary thereby the mis-folded RNAs interacts with another cavity. The uniqueness of these RNA species alongside with its relevance to autoimmune disease still needs to be explored .
E.coli, serves as the preferred choice of vector used towards cloning recombinant protein, as it can produce optimal quantity, easy to grow, can be easily handled and manipulated, easier steps of purification of products. These factors makes it superlative and preferred choice. This work attempts to clone TROVE2 transcript variant from the pBluescript Plasmid.
The pBluescript phagemids refers to plasmids having phage origin. It is the best serving cloning vector which is designed towards simplifying the most commonly utilized cloning as well as sequencing protocols, that included the nested deletions construction towards DNA sequencing, as well as RNA transcripts generation of both in vitro as well as site-specific mutagenesis featuring gene mapping. The pBluescript has extensive polylinker containing 21 of unique restriction enzyme recognition sites. the polylinker are Flanked with T7 as well as T3 RNA polymerase promoters which are used towards in vitro RNA synthesis. The promoter’s choice serves towards initiating transcription which indeed determines the strand to be cloned by insertion into the polylinker that will be indeed transcribed.
The transcripts were isolated from both the tyrosine kinase inhibitor treated as well as the untreated KCL22 cells. For successful cloning, RNA was extracted subsequently cDNA was subjected to amplification using PCR. The plasmids were purified, digested and the amplified cDNA was cloned and transformed into vector. Human leukemic cell line (KCL22) were selected for this study.
The Kcl22 cell line is being used extensively owing to its use as exceedingly sensitive target in vitro for various assays. Cultures of ATCC stock exhibit sensitivity towards assessment of human natural killer activity. They are Chronic myelogenous leukemia, BCR-ABL1 positive cell line.These are hematopoietic stem cells having capacity to differentiate to form erythroid, megakaryocytic lineages. The entire RNA content was extracted from KCL22 cell lines, and it was subjected to reverse transcription. The obtained CDNA quality was affirmed by real time qPCR. Amplified product of TROVE2 transcript was cloned into vector subsequent to digestion with selected restriction digestion with pst1 and sgf1. Then, it was ligated by using Bioline QS ligase enzyme. Transformation was carried by heat shock method. Blue white selection strategy was employed to select the transformed colonies. Colony PCR was performed for ascertaining the proper cloning. Agarose gel electrophoresis was employed to separate DNA fragments and analysed fragments cross checked for ascertainment.
Tissue culture of KCL22 cells were grown as per routine standard tissue culture conditions. Treated KCL22 cellular samples were grown with media being supplemented with tyrosine kinase inhibitor( TKI) drug for a period of 72h whereas untreated cells were indeed cultured in medium devoid of TKI drug. The morphology of KCL22 cells were subsequently observed by inverted microscope. Viability as well as distribution of size was observed using automated cell counter of BioRad T make.
The average percentage has relatively dropped with treated cells (Table 1A and B).
Table 1A and B:
Preparation of plasmid from E.coli: Preparation of pBluescript SK+ plasmid in E.coli was carried over by growing the E.coli cells with LB media supplemented with ampicillin antibiotic. pBluescript SK+ plasmids were purified using ‘plasmid purification’ spin column kit. The concentration of purified plasmid was ascertained using Invitrogen Qubit flourometer by using Quant-IT dsDNA BR dye. Results indicate that the fractions were pure.
Subsequently, it was subjected to restriction enzyme digestion with restriction digestion buffer. Then treatment with thermo sensitive alkaline phosphatase was carried out to prevent self-ligation. The digestion was ascertained by agarose gel electrophoresis for detection of complete digestion of plasmid (figure 1).
Figure 1: The third result belong to student K shows that the different between uncut plasmid and digested plasmid.
Using commercial spin column purification kit , the total RNA was purified from the cells. Then, by using a standard denaturing agarose gel electrophoresis the quality of an RNA sample was ascertained. The RQI value (RNA Quality Indicator) was estimated value of how ‘intact’ the RNA is in a sample. This value is again being determined by the 28s – 18s ratio of the RNA sample. It will be compared with gel images.
Qubit RNA analysis – RNA concentration only
Student letter RNA [μg/mL] Cell type
A 254.0 KCL22
B 112.4 K562
C 432.0 K562
D 426.0 K562
E 982.0 K562
F 906.0 KCL22
G 137.6 KCL22
H 339.0 KCL22
I 584.0 K562
J 654.0 K562
(K) 402.0 KCL22
L 305.6 KCL22
M 187.0 KCL22
N 49.0 K562
O 464.0 K562
P 394.0 KCL22
Z 102.0 KCL22
The student K sample extracted 402.0 μg/mL of RNA relative to average of 395.92 μg/mL. The quantity of RNA extraction reveals the effective handling, protocol effectiveness.
The 28S and18S RNA bands were observed on the gel as it was expected, at approximately 5000 bases length for the 28S rRNA, and approximately 1908 bases for the18S rRNA.The ratio between 28S/18S is 2:1.RNA concentration in the TKI treated μg/ml sample was 332.0 and the RQI value is was10.
Preparation of cDNA:
cDNA was extracted from KCL22 cells and amplified by qPCR. Expression levels were analyzed by CFX manager determined the untreated cells from treated cells. A set of real time qPCR reactions were performed in order to determine the quality of the synthesized cDNA. Four specific primers were used to amplify TROVE2 transcript variant.
μls of sample required to give 1μg of total RNA in cDNA synthesis reaction μl of qPCR grade water required to to cDNA synthesis reaction mix Total μl of Reaction buffer master mix Total
A 3.9 10 14.0 6.0 20.0
B 8.9 5 14.0 6.0 20.0
C 2.3 12 14.0 6.0 20.0
D 2.3 12 14.0 6.0 20.0
E 1.0 13 14.0 6.0 20.0
F 1.1 13 14.0 6.0 20.0
G 7.3 7 14.0 6.0 20.0
H 2.9 11 14.0 6.0 20.0
I 1.7 12 14.0 6.0 20.0
J 1.5 12 14.0 6.0 20.0
K 2.5 12 14.0 6.0 20.0
L 3.3 11 14.0 6.0 20.0
M 5.3 9 14.0 6.0 20.0
N 2.0 12 14.0 6.0 20.0
O 2.2 12 14.0 6.0 20.0
P 14.0 0 14.0 6.0 20.0
Z 9.8 4 14.0 6.0 20.0
Student K has used 2.5 ul of sample for RNA concentration of 1 ug for CDNA synthesis.
The quality of cDNA synthesis:
Real-Time PCR thermal cycles used to synthesis complementary DNA. was synthesized from RNA in the process of RT-PCR and BioRad iScript Advanced cDNA synthesis kit.
cDNA was synthesized from the RNA by reverse transcription PCR (RT-PCR) by virtue of combination of reverse transcriptase enzyme, along with specific primers. Such primers along with the enzymes helps to transcribe all of the RNA transcript into cDNA. However, all of these transcripts variants (TROVE2,TROVE2 5’ASS, and TROVE2 wild-type) must be amplified during the RT-PCR. Subsequent to cDNA synthesis, a quantitative PCR (qPCR) was carried out by transcript variants with specific primers in addition to contaminating genomic DNA (gDNA) primer and SYBR green mix towards determining the quality of cDNA.
The chart above shows the qPCR result for the student K sample.
The normal average percentage has moderately dropped pertaining to the treated cells. This is indeed in correlation with reports of USFDA approved tyrosine kinase inhibitors nilotinib dasatinib, matinib, which act as first-line of treatment towards treating CML in chronic-phase (CML-CP). For student K the untreated cellular viabily is 100% and it had declined with treated cells remarking that the cell lines are being inhibited by TKI treatment.
The pBluescript SK+ plasmid were prepared in E.coli successfully while the purity of plasmid was ascertained by virtue of qubit analysis. The obtained results were in accordance with probability enlightening that the method used was finest towards extracting plasmid in its purest form.
Figure 2: Column five shows the digested plasmid with expected size.
The total RNA was purified by commercial spin column purification kit. The RQI value (RNA Quality Indicator) reflects the ‘intactness’ of the RNA within the sample. This value is determined by the ratio of 28s – 18s RNA in the sample provided. It was correlated with gel images. The values obtained were within expected range. Sample K had extracted 402.0ug/ml of RNA from KCL22 cells in range with the class average ( 394 ug/ml) extraction reflecting the efficiency of extraction.
cDNA being extracted from KCL22 cells were then amplified by qPCR. A set of real time qPCR reactions were carried over towards determining the synthesized cDNA quality. Four of specific primers were utilized for amplifying the variant of TROVE2 transcript and the results implicate that Student K has utilized 2.5 ul of sample for RNA concentration of 1 ug for CDNA synthesis. The primers transcripts variants (TROVE2,TROVE2 5’ASS, and TROVE2 wild-type) were utilized for amplification during the RT-PCR. The quantity of TROVE2 transcript amplified were optimal enough to carry over further transformation Figure 2.
Colony PCR products on 1% agarose gel to detect E.coli cells that contain recombinant plasmids with gene of interest.
Figure 3: The image shows Colony PCR products on 1% agarose gel A) It characterizes the 4 blue E.coli colonies analyzed by PCR in order to confirm there is no TROVE2 insert. Lane 1 represents Hyper- Ladder 1kb. Lane 2,3,4 and 5 displays the colonies that obtain from student K plates. All of the colonies were negative colony PCR as the insert was not detected. which indicates these colonies do not have the insert gene. B) it characterizes the 3 unlike white colonies gotten from other student’s plates (M, I and G) and show a successful clone for TROVE2 insert in M student colony.
The Cloning of TROVE2 transcript variant 1 which extracted from KCL22 cell line(CML cells) into E.Coli with pBluescript plasmid. pBluescript plasmid was extracted from E.coli, digested by using specific restriction enzymes (EcoR1) and purified . The detecting of phenotype, counting and viability of chronic myeloid leukemia (CML) cell line (KCL22), as well extracting the RNA from KCL22 cells. Complementary DNA was synthesized from total RNA and perform PCR amplification of TROVE2 (Gene of interest).
Moreover, digest gene of interest (GOI) from KCL22 cell and approximating the molar ratio of the digested plasmid and the GOI. As well, do transformation procedures to insert the gene into the plasmid.
Colony PCR used to detect the recombinant plasmid after Screening different bacterial colonies.
After incubation of ligation reactions for one day and then spread them on plates. Unfortunately, there were no results on the ligation plates for most of the students including student K, but there were colonies on control plates. However, there were some students (M, I and G) who got colonies grew on their ligation plates but only one student (M) got result on agarose gel after we have done PCR colony.
The growth on (M) ligation plate showing that the transformation was successful as well as the cloning of TROVE 2 variant 1 which seen on agarose gel electrophoresis (Figure 3). The next step that we should done is sequencing for the insert to be sure it’s on open frame then create copies of that product which allow us to study the gene of interest. The confirmed result after published allow scientists to make experiments on the gene which could help in CML treatment.
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