Wednesday, June 30, 2021

Classic model HT-29 cell line-a unique model for colorectal cancer research! Ubigene Biosciences

 Background


The HT-29 cell line was isolated from a 44-year-old woman in 1964 by two scientists, Fogh and Trempe. HT-29 cell line have the characteristics of mature intestinal cells. When HT-29 exists in different culture conditions or inducers, the cells will show different differentiation pathways. Therefore, HT-29 cells are a model for the study of the molecular mechanism of intestinal cell differentiation. HT-29 cells are popular in research. Ubigene also has made a deep study on HT-29 cells’ culture system and gene knockout system, and Ubigene can achieve 100% protein knockout in HT-29 cells.

 Applications of HT-29 cell line


So far, HT-29 cells are used in various basic researches and biomedical studies, involving lung cancer proliferation, corresponding inhibitor research, tumor formation, metastasis, and so on.

(1) Studying new synthetic drugs in the biomedical field: For example:HT-29 cells were used as a model to study the cytotoxic mechanism of polyphenol stabilized colloidal gold nanoparticles prepared from the Abutilon Indicum leaves on colon cancer cells (HT-29)[1]

(2) Research of food industrial residues:For example, using fruit juice production residues as natural anti colon cancer drugs, studies have found that fruit juice production residues can effectively inhibit the cell proliferation of HT-29 cells and induce cell cycle arrest at different arrest points G1, G2 / M and S[2],This research has a certain role in promoting the research and development of anti colon cancer drugs.

(3) Completing the molecular mechanism of colorectal cancer formation:For example, prolyl 4-hydroxylase, βPeptide (p4HB) is related to the occurrence of colon cancer. The release of p4HB knockdown reduced the activation of the signal converter and transcription activator 3 (STAT3), and promoted the accumulation of ROS, and then induced apoptosis of HT-29 cells[3]

(4) Study on the effect of natural fermentation products on colon cancer cells:For example, it was found that after the treatment of a natural fermentation product Lactobacillus Acidophilus CICC 6074 S-layer protein, there were chromatin condensation, nuclear fragmentation, vacuoles, and other apoptotic characteristics in the cells. This protein may induce HT-29 cell apoptosis through the death receptor apoptosis pathway and mitochondrial pathway, and inhibit cell invasion by inhibiting the synthesis of the PI3K/AKT pathway and FasL pathway[4]

The Application of CRISPR/Cas9 Technology in HT-29 Cell

Line


CRISPR/Cas9 technology is a highly specific method for gene editing. This technology can induce sequence deletion, insertion, and point mutation in the cell. dCas9 can block transcription and inhibit gene expression. CRISPR/Cas9 technology has also been widely used in HT-29 cells. For example, p38γ is a new cyclin-dependent kinase (CDK), it found that in HT-29 cells and primary human colon cancer cells, shRNA induced p38γ silencing or CRISPR/Cas9 mediated p38γ knockout inhibited cell growth, proliferation, and migration, and significantly induced cell apoptosis[7]


The following is a case of CRISPR/Cas9 technology in colorectal cancer cell resistance mechanism research[8],which is convenient for you to develop new ideas.Drug therapy is a way of colorectal cancer treatment, but continuous use of drugs can cause resistance of colorectal cancer cells. 5-Fluorouracil (5-FU) is an artificial nucleotide analog. Since the 1950s, it has been the main drug for chemotherapy in CRC patients, mainly by inhibiting thymidylate synthase and incorporating its metabolites into DNA, leading to cell death. In addition, oxaliplatin can also be used in the treatment of colorectal cancer patients, which belongs to the third generation of platinum drugs and can form platinum DNA complex to induce cytotoxicity.


Jinming Yu et.al established HT-29 and SW480 drug-resistant cell lines by long-term treatment with 5-FU and oxaliplatin, respectively.Western blot analysis showed that REV7( Translation synthesis polymerase ζ( POL ζ) key components of) was up-regulated in 5-FU and oxaliplatin resistant CRC cells (Fig. 1. A-D), REV7 in HT-29 and SW480 also increased in a dose-dependent manner (Fig. 1. E-H). But the interesting thing is that there was no significant difference in the expression of REV7 mRNA after treatment, which means that the increase of REV7 protein was not caused by the change of transcription level.


Fig 1.


Jinming Yu et al. used CRISPR/Cas9 technology to specifically target the REV7 gene. Under drug treatment, REV7 was almost not expressed in REV7 deficient cell lines (Fig. 2. a-b). Plasma-based TLS efficiency analysis showed that the deletion of REV7 significantly inhibited the TLS (translesion synthesis) efficiency in drug-resistant HT-29 cells, and this phenomenon was alleviated after the supplement of REV7 (Fig. 2. c-d).


Fig 2.


It was found that the expression of REV7 did not affect the tumor volume without drug stimulation in mice xenotransplantation of drug-resistant cell lines, indicating that REV7 did not affect the occurrence of colorectal cancer. When given drug stimulation, the absence of REV7 significantly inhibited tumor growth, but there was no significant difference in individual weight (Fig. 3).


Fig 3.


In conclusion, HT-29 cell line as a classic cancer cell model are very important for the study of colorectal cancer, and CRISPR/Cas9 also plays a very important role in molecular research methods. So far, Ubigene has used CRISPR-U ™ technology to successfully modified genes from more than 100 cell lines, including the HT-29 cell line. And Ubigene provides gene-editing services worldwide. Now Ubigene has time-limited discount of all gene-editing services: KO cell line service, as low as 3780 USDKI/Point mutation cell line service, as low as 8480 USDgRNA plasmids, as low as 80 USD



References:

[1]Sadras Sudha Rani, et al. Polyphenol stabilized colloidal gold nanoparticles from Abutilon indicum leaf extract induce apoptosis in HT-29 colon cancer cells[J]. Colloids Surf B Biointerfaces, 2016, 143: 499-510.

[2]Catarina M.M.Duarte, et al. Evaluation of Opuntia spp. derived products as antiproliferative agents in human colon cancer cell line (HT29)[J]. Food Research International, 2013, 54: 892-901.

[3]Zhang Qilin, et al. P4HB knockdown induces human HT29 colon cancer cell apoptosis through the generation of reactive oxygen species and inactivation of STAT3 signaling[J] .Mol Med Rep, 2019, 19: 231-237.

[4]Yuxing Guo, et al. Effect of Lactobacillus acidophilus CICC 6074 S-Layer Protein on Colon Cancer HT-29 Cell Proliferation and Apoptosis[J]. J Agric Food Chem, 2020, 68: 2639-2647.

[5]Yaguang Xi, et al. CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo[J]. Sci Rep, 2016, 6: 22312.

[6]Jinke Wang,et al. Cancer gene therapy by NF-κB-activated cancer cell-specific expression of CRISPR/Cas9 targeting to telomere. doi: https://doi.org/10.1101/553099

[7]Yiou Cao, et al. Targeting p38γ to inhibit human colorectal cancer cell progression[J].Biochem Biophys Res Commun. 2019 Sep 10;517(1):172-179. doi: 10.1016/j.bbrc.2019.07.038.

[8]Jinming Yu, et al. Targeting REV7 effectively reverses 5-FU and oxaliplatin resistance in colorectal cancer[J].Cancer Cell Int, 2020, 20: 580.

Tuesday, June 29, 2021

You must check out this highly-recommended gRNA design tool | Ubigene


As long as you know a little bit about CRISPR/cas9 technology, you must hear that during CRISPR/cas9 experiments, the design of the gRNA will largely affect the success rate of the project. The cas9 nuclease, under the guide of gRNA, targets the gene of interest and generates a double stranded DNA break (DSB). Insertions and deletions (indels) arise as a result of random repair of DSBs, causing frameshift mutations that result in premature stop codons, this process can achieve the purpose of gene knockout. But in most cases, many people often face difficulties at the stage of gRNA design. So this week we’ve also received some questions about gRNA design and selection, let’s start our discussion:

Q1:

While we design the gRNA to KO the target gene using CRISDPR/Cas9 technology, should I use single gRNA or dual gRNA? What is the difference between them?

A:

In theory, a single gRNA can target only 1 site of a gene, and the number of transcripts that can be knocked out is limited. While a dual gRNA can largely solve the problems, and can also KO a larger fragment of gene, so the KO effect of the dual gRNA vector will be better than that of the single gRNA vector. But in practice, designing an apporpriate strategy depends on specific cases and analysis.

To get the desired experimental results, we currently use the Red Cotton™ CRISPR gene editing system (Go) for gene KO strategy design. This powerful system integrates experimental data from more than 5000 successful cases of gene editing with several mainstream gene databases and it provides 3 different gene KO strategies for free by just entering the gene name. It can reduce our experimental trial and error costs.

Q2:

What are the principles when designing the gRNA? Could you recommend a useful gRNA design tool?

A:

You should take many factors into consideration when designing the gRNA, here are the three most important rules you should consider:

  • The sequence specificity of the gRNA should be strong enough
  • gRNA sequences to try to cover as many transcripts as possible
  • Avoid target regions that encode amino acids too close to the N- or C-terminus of the protein

But in fact we usually do the design of gRNAs with the help of the gRNA design tool. A good gRNA design tool can improve efficiency and reduce the trial and error cost. I recommend using our Red CottonTM system - gRNA design tool with the help of the sgRNA design website by Zhang’s lab. But just a reminder, it is not the higher score of gRNAs, the better its KO effect. Before you make the decision, you can have a comprehensive knowledge of the target gene using our Red Cotton system.

Q3:

 How can I improve the targeting efficiency of gRNAs?

A:

How to improve the targeting efficiency of gRNAs is a matter of great concern to every researcher working on CRISPR/cas9 technology. Either sequence optimization of the gRNA, changing the delivery method of the gRNA vector, or increasing the number of gRNAs used, etc., can improve the targeting efficiency of gRNAs to some extent.

Hence, there are many ways to improve the targeting efficiency by optimization according to the actual situations. Welcome to use our free Red Cotton™ CRISPR gene editing system (Go) or feel free to contact us if you need help!

Above are the Q&A of this week. If you have any similar questions, feel free to contact us now!

 

Ubigene makes genome editing easier!

Monday, June 28, 2021

Combination of HCT116 cells and CRISPR/Cas9: A perfect group for colorectal cancer treatment research | Ubigene

 


 1. Cell information and applications

1.1 Cell information


HCT 116 was isolated from a 48 year old male patient with colorectal cancer in 1979 by M. brattain et al. The cells can form clones in semisolid agarose medium and are tumorigenic in athymic nude mice, forming epithelial-like tumors. So HCT 116 cell line is one of epithelial-like adherent tumor cell lines.


1.2 Cell applications and prospects


(1) To explore the mechanisms by which drugs affect colorectal cancer cell proliferation, migration and invasion. For example: PPARα plays an important role in the migratory activity and the expression of cyp2s1 and CYP1B1 of leukocyte treated HCT116 cells [2];

(2) To explore mechanisms of action in vitro by which drugs affect colorectal cancer cell growth, such as apoptosis and cell cycle alterations. For example: Raddeanin A regulates apoptosis and cycle arrest in human HCT116 cells through the PI3K/Akt pathway [3];

(3)To explore the sensitivity and resistance of drugs to colorectal cancer treatment. For example: Doxorubicin upregulates PD-L1 by inhibiting mir-140 expression in HCT116 cells, thereby rendering tumor cells being resistant to the drug [4];

(4)Development of new cancer-related signaling pathways to provide guidance for clinical treatment of colorectal cancer. For example, to investigate the significance of Notch and Wnt signaling pathways to drug resistance in colorectal cancer cell line HCT116, etc. [5];

(5)Developing new approaches of LncRNA and miRNAs in colorectal cancer treatment. For example: in HCT116 cells, long non-coding RNA of YWHAE competes with miR-323a-3p and miR-532-5p by activating K-Ras/Erk1/2 and PI3K/Akt signaling pathways. It provides a new target site for colorectal cancer treatment. [6];

 2.The application of CRISPR/Cas9 technology in HCT116

As mentioned above, HCT116 cells have been of great research value in the studies on various mechanisms of colon cancer, and the most basic research ideas generally start at the molecular level, that is, gene or protein. Thus, CRISPR/Cas9 technology has emerged in many research topics, and it is nonetheless an irreplaceable advantage in studying the functions of single carcinogenesis gene.


Some researches found that Trpm4 is highly expressed in human colorectal cancer and it seems to be associated with colorectal cancer cell proliferation, cell cycle and invasion. It was found that both tumor cell migration and invasion were indeed reduced by knocking out Trpm4 using CRISPR/Cas9 technology in HCT116 cells, also accompanied by cell cycle alterations [7];


As well as Cell reports that using CRISPR/Cas9 technology, replacing wild-type KRAS with mutant forms rendered the heterozygous mutant HCT116 cells more sensitive to drug treatment [8];


Moreover, by using CRISPR/Cas9 to knockout Tks4 in HCT116 cells, the cells exhibited significant epithelial mesenchymal transition, increased cell motility, and decreased cell-cell adhesion. Therefore found that the Tks4 gene plays an important role in EMT regulation and tumor development [9];


Researchers also found that the CTC1L1142H mutation resulted in impaired telomerase maintenance, by using CRISPR/Cas9 technology to mutate CTC1 in HCT116 cells. It confirmed that the CTC1:STN1 interaction was required for the inhibition of telomerase activity [10].


Therefore, there is no doubt among researchers for the favor of CRISPR/Cas9 technology. Ubigene, by providing the CRISPR/Cas9 technology services, has solved the experimental difficulties for many researchers, such as incomplete cell knockout, unstable cell knock-in, etc.. With our services, more and more researchers can achieve their scientific research objectives smoothly.


 3. Case Studies

3.1 Poing mutation[11]


Hiroyuki Kato et al. using CRISPR/Cas9 technology introduced the mutations on UTX gene in HCT16 cells, which is G137V and G137VΔ138. They found that the wild-type UTX, which was originally expressed in the nucleus, greatly decreased in the nucleus, whereas increased in the cytoplasm in both mutant cells. As shown in Figure A to C: A, B were the immunofluorescence figures, C was the Western blot results. The results of co-immunoprecipitation are shown in fig.d, it is a novel regulatory mechanism of UTX. Also, the article also further revealed the importance of UTX interaction with MLL3/4 complex in cancer formation (ASH2L, PTIP and PA1 are components of MLL3/4 complex) .



3.2 Knockout [12]


CRISPR/Cas9 gene-editing technology is used to obtain HCT116 cell line with DAPK1 deletion by Sara Steinmann et al, and finally revealed the effect of DAPK1 on the invasion of colorectal cancer.


After Western Blot verification, three DAPK1 gene knockout monoclonal cells (Fig. A) were obtained by Sara Steinmann et al. Immunofluorescence assays detected pERK1/2 mainly in the wild-type HCT116 cytoplasm. However, in all three knockout cells, pERK1/2 was significantly expressed in the nucleus. (Fig. B)


Because the chorionic allantoic membrane model (CAM) experiment is a classic in vivo model of angiogenesis, the researchers transplanted DAPK1 knockout clone and wild- type HCT116 cells to chicken CAM, and cultured in eggs for 5 days. It was found that the absence of DAPK1 led to the change of growth pattern and enhancement of tumor bud in CAM ( Fig. C)


In addition, the team used a rat brain 3D model in vitro to find that tumor cells had more proliferation in chicken embryonic organs, and the invasion ability was also enhanced. DAPK deficient HCT116 cells showed more diffuse tumor cells and preferentially accumulated in the liver, heart, and brain of the chicken embryo (Fig. D). Finally, the researchers found that DAPK1-ERK1 signaling pathway is involved in the metastasis of CRC (Fig. E).





3.3 Knock-in [13]


BAX is one of the members of pro-apoptotic Bcl-2 gene family, which plays an important role in mitochondria dependent apoptosis initiation. R Peng et al have knocked in five mutation sites in BAX-KO HCT116 cells, which are located in the BAX gene and interact with other members of Bcl-2.


It has been reported that BAX-KO HCT116 cells do not undergo apoptosis by the stimulation of sulindac, a steroidal drug. R Peng et al found that knock in WT BAX in BAX-KO HCT116 cells can restore sulindac, and can restore HCT116 apoptosis caused by TRAIL. After knocking in the K21E and D33A mutations, BAX mediated apoptosis was completely restored; Knocking-in D68R and S184V mutations only partially recovered, while knocking in L70A / D71A mutation caused less apoptosis.



In this study, the target gene in the target cell is knocked out, and then knock in the target gene containing the mutation site and WT target gene (contrast), then we can clearly understand the specific site relationship between the protein expressed by the target gene and other proteins interacting with it, It is a good method to verify whether the predicted sites obtained by bioinformatics analysis really play a role in protein-protein interactionUbigene also provides the corresponding service convenience for scientific researchers. While enjoying the gene KI cell line service, free knockout cells can be obtained which meets the various research needs.

Ubigene CRISPR-UTM technology with 5000 successful experiences makes our gene-editing services more trustworthy! We have promotions for gene-editing services now: KO cell line services as low as 3780 USD KI/PM cell line services as low as 8480 USDgRNA plasmids as low as 80 USD

Reference:

[1]Bray, Freddie et al. “Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.” CA: a cancer journal for cliniciansvol. 68,6 (2018): 394-424. doi:10.3322/caac.21492

[2]Khor CY, Khoo BY. PPARα plays an important role in the migration activity, and the expression of CYP2S1 and CYP1B1 in chrysin-treated HCT116 cells. Biotechnol Lett. 2020;42(8):1581-1595. doi:10.1007/s10529-020-02904-

[3]Meng C, Teng Y, Jiang X. Raddeanin A Induces Apoptosis and Cycle Arrest in Human HCT116 Cells through PI3K/AKT Pathway Regulation In Vitro and In Vivo. Evid Based Complement Alternat Med. 2019;2019:7457105. Published 2019 May 26. doi:10.1155/2019/7457105

[4]Naba NM, Tolay N, Erman B, Sayi Yazgan A. Doxorubicin inhibits miR-140 expression and upregulates PD-L1 expression in HCT116 cells, opposite to its effects on MDA-MB-231 cells. Turk J Biol. 2020;44(1):15-23. Published 2020 Feb 17. doi:10.3906/biy-1909-12

[5]Kukcinaviciute, Egle et al. “Significance of Notch and Wnt signaling for chemoresistance of colorectal cancer cells HCT116.” Journal of cellular biochemistry vol. 119,7 (2018): 5913-5920. doi:10.1002/jcb.26783

[6]Bjeije, Hassan et al. “YWHAE long non-coding RNA competes with miR-323a-3p and miR-532-5p through activating K-Ras/Erk1/2 and PI3K/Akt signaling pathways in HCT116 cells.” Human molecular genetics vol. 28,19 (2019): 3219-3231. doi:10.1093/hmg/ddz146

[7]Kappel, Sven et al. “TRPM4 is highly expressed in human colorectal tumor buds and contributes to proliferation, cell cycle, and invasion of colorectal cancer cells.” Molecular oncology vol. 13,11 (2019): 2393-2405. doi:10.1002/1878-0261.12566

[8]Szeder, Bálint et al. “Absence of the Tks4 Scaffold Protein Induces Epithelial-Mesenchymal Transition-Like Changes in Human Colon Cancer Cells.” Cells vol. 8,11 1343. 29 Oct. 2019, doi:10.3390/cells8111343

[9]Burgess, Michael R et al. “KRAS Allelic Imbalance Enhances Fitness and Modulates MAP Kinase Dependence in Cancer.” Cell vol. 168,5 (2017): 817-829.e15. doi:10.1016/j.cell.2017.01.020

[10]Gu, Peili et al. “CTC1-STN1 coordinates G- and C-strand synthesis to regulate telomere length.” Aging cell vol. 17,4 (2018): e12783. doi:10.1111/acel.12783

[11]Kato, Hiroyuki et al. “Cancer-derived UTX TPR mutations G137V and D336G impair interaction with MLL3/4 complexes and affect UTX subcellular localization.” Oncogene vol. 39,16 (2020): 3322-3335. doi:10.1038/s41388-020-1218-3

[12]Steinmann, Sara et al. “DAPK1 loss triggers tumor invasion in colorectal tumor cells.” Cell death & disease vol. 10,12 895. 26 Nov. 2019, doi:10.1038/s41419-019-2122-z

[13]Peng, R et al. “Targeting Bax interaction sites reveals that only homo-oligomerization sites are essential for its activation.” Cell death and differentiation vol. 20,5 (2013): 744-54. doi:10.1038/cdd.2013.4

Wednesday, June 16, 2021

Will CRISPR/Cas9 technology lead to new development of cell genetic modification? Ubigene

 

Since the world’s first CRISPR/Cas9 clinical trial was carried out in West China Hospital in 2016, gene therapy for cancer has made many gratifying breakthroughs with the help of CRISPR/Cas9, and the breakthrough of CRISPR/Cas9 also attracted everyone's attention. Ubigene has been committed to CRISPR/Cas9 system in cell gene-editing experiments. We are ahead of other companies and promise to guarantee 100% no protein residue in 5000 gene knockout experiments. We also pay attention to the progress of CRISPR/Cas9 system in the field of cancer. Here are a few cases of cancer research using CRISPR/Cas9 system (the following cells are all successful cases of Ubigene):

Gene editing of Huh-7 cell line——Boost coronavirus, drug metabolism and cancer research

Huh-7 cell line was established in 1982 by Nakabayshi, H. and Sato, J. it is epithelioid and highly heterogeneous. In order to overcome the shortcomings of primary hepatocytes in drug metabolism research, a CRISPR/Cas9 gene modified human hepatocyte line was developed,which studies the effects of gene variation on drug metabolism. Cancer stem cells (CSCs) are closely related to the occurrence and metastasis of cancer. They have the ability of self-renewal and unlimited proliferation, and are the key factors in the development of cancer. Researchers found that the expression of androgen receptor was very high in liver cancer tissues, and was related to Nanog. CRISPR/Cas9 technology was used to knock GFP into Huh-7 cells Nanog, revealing the gender difference in incidence rate of hepatocellular carcinoma, and providing a possible way for the suppression of axons in liver cancer treatment. 

click here for the full article

Gene editing CT26.WT cell line——A magic way for colon cancer research and treatment

CT26.WT cell is an invasive mouse colon cancer cell line. By using CRISPR/Cas9 system to edit gene in this cell line, we can generate single or multiple gene knockout, mutation correction or reporter gene insertion transgenic cells. It has a wide range of applications in the study of cancer markers, revealing the mechanism of drug resistance, cancer treatment, cell death and other fields. The researchers used CRISPR/Cas9 system to knock out ATG7 in mouse melanoma cell line B16F10, mouse colon cancer cell line MC38 and mouse colon adenocarcinoma cell line CT26 to study the role of autophagy in the proliferation of mouse cancer cells, and finally revealed the effect of autophagy destruction on immune reactive tumors.

click here for the full article


H1299 cells——A tool of studying CRISPR gene therapy for cancer mutation

As an immortalized cell line, H1299 can divide infinitely. The unique feature of this cell line is the lack of P53 protein expression. As a disease model, H1299 cell line plays a very important role in understanding the basic biology of disease, understanding how mutations affect drug response or drug resistance, understanding drug reactivity, target recognition, verifying the mechanism of differences, and even stratification of patients. In one study, researchers used CRISPR/Cas9 technology to construct BCAR1 gene knockout on lung adenocarcinoma cell lines (ncl-h1975 and ncl-h1299), and showing that BCAR1 promoted the proliferation and cell growth of lung adenocarcinoma by up regulating POLR2A.

click here for the full article

Ubigene has been working to allow CRISPR/Cas9 technology to be better developed and applied, and our independently developed CRISPR-U™ Technology is 10-20 times more efficient than traditional gene editing, also the breakthrough patented technology for gene modification in vitro and in vivo. Moreover, Ubigene uses CRISPR-U™ to successfully edit genes from more than 100 cell lines. Our leading technology with abundant experience makes our gene editing services more trustworthy!

Wednesday, June 9, 2021

Point mutation cell line generation--Ubigene Weekly Q&A

 

Nowadays, many researches indicate that CRISPR/Cas9 is becoming the optimal tool to construct the models of human diseases, such as human pluripotent stem cells and tumor cell lines. With modern sequencing, CRISPR/Cas9 can introduce precise point mutations (homozygous or heterozygous) in various cell lines and enables to construct human disease models. This is highly advantageous for studying both the therapy and the underlying genome of the diseases.

 

We also received a few questions this week about introduction of point mutation in cells using CRISPR/Cas9. Below are the Q&As for this week.

 

Q1 How can I use CRISPR/Cas9 technology to study gene point mutation cell lines?

A

The specific mutations can be achieved by CRISPR/Cas9 efficient introduction of targeted double-strand breaks (DSBs), repaired using DNA repair donor (e.g., single-stranded oligo DNA nucleotide, ssODN) under homology-directed repair (HDR) system. Importantly, with the KI cassette based on the CRISPR/Cas9 system, we can also selectively introduce mono-allelic and dual-allelic sequence changes, which are heterozygous and homozygous point mutations to construct pathogenic disease models.

 

Homozygous point mutations require gRNAs to target positions close to the target mutation site, whereas heterozygous point mutations can be achieved by distance-dependent incorporation of suboptimal mutations or by using mixed repair templates.

 

So, the introduction of point mutations in cell lines mediated by CRISPR/Cas9 technology is very useful for the studies on the gain of function or loss of function.

 

Q2 Hi there, I want to construct a gene point mutated Hep-G2 cell line. For reference, could you tell me the estimated timeline for the whole experiment?

A

For endogenous point mutation, it’s a bit challenging to introduce in Hep-G2 because the proliferation rate for Hep-G2 is relatively slow and the single-cell colony formation rate is relatively lower than other common cell line models. If everything goes smoothly, the whole process takes about 3-4 months. Because it is difficult and time consuming, you could consider outsourcing the experiment.

 

Ubigene is experienced in successfully modifying Hep-G2 cell line and if you order point mutation cell line generation services from Ubigene, we will give you the KO cell line for free, making your experiment and researches more convenient.

 

Q3 Hi there, I want to introduce a point mutation (change of an amino acid) in the target gene using CRISPR/Cas9 technology, and I want to do cell transfection and single-cell clone isolation in my own lab. I have searched many articles and checked some information from addgene, found that I need to construct a donor DNA to achieve the point mutation in the cells. How to design the constructs and how much budget is generally required?

A

The donor DNA is not as complicated as you think to construct. A donor comprises a sequence carrying the mutation and upstream and downstream sequences (each about 1-2Kb long) for the purpose of homologous recombination. Or you could also use oligo which has shorter upstream and downstream sequences (about 100-200bp) to achieve the point mutation.

For the question of budget, the regular price for a set of point mutation constructs and oligo is about 770 USD in Ubigene.

Above are the Q&As of the week.

Feel free to contact us if you have any similar questions.

 

Ubigene makes genome editing easier!

[Research highlight]A new target for human endometrial cancer treatment |Ubigene

  Human endometrial cancer (EC) is one of the malignant tumors of the female reproductive tract . With the increase of aging populations, ob...