Knocking-in Protein Tags Into The C-terminus Of KLHL21 In HEK293T Cells

The KLHL(Kelch-like)family have one POZ/BTB(bric-a-brac tramtrack and broad complex)domain,occasionally a BACK domain,and several repeats of the Kelch domain.The KLHL family is conserved throughout evolution,suggesting that it may play an important physiological function in the body.KLHL proteins are known to be involved in the ubiquitination process through cullin E3 ligases,but the specific roles have not been elucidated.Functions observed to be involve a variety of celluar processes such as inflammatory responses,oxidative stress responses,cytokinesis,embryonic development,lymphangiogenesis and others remain unclear.The KLHL21 gene is clearly a member of KLHLfamily,have one POZ/BTB domain,a BACK domain and five Kelch domain.In 2009,Maerki S was first reported that KLHL21 can combine with the Cullin3 protein forms the functional E3 ubiquitin ligase and catalytic the ubiquitination of protein kinase Aurora B,while KLHL21 is necessary for cytokinesis and regulates translocation of the CPC(Chromosomal passenger complex)from chromosomes to the spindle midzone in anaohase,siRNA against KLHL21 led to severe defects during mitosis and failed to complete cytokinesis.From the latest research,Maerki S also reported that KLHL21 localizes to FA structures preferentially at the leading edge,and in complex with Cul3,ubiquitylates EB1 protein and proceed to regulate cortical dynamics during cell migration.The I?B kinase(IKK)complex is composed of two catalytic subunits(IKK?,IKK?)and a regulatory subunit(NEMO,also known as IKK?),its activation is the key event in canonical NF-?B signaling.Our preliminary work identified that KLHL21 as a novel negative regulator of IKK? can specifically binds to the KD domain of IKK? through its Kelch domains and inhibits IKK? activation,and then differential regulate the expression of NF-?B target genes independently of its E3 ubiquitin ligase activity.In previous work,we have employed some KLHL21 antibody from differential biotech firms,however none of those can afford our experiment on account of the poorly sensitivity and specificity.In this study we attempt to knockin protein tags into the C-terminus of KLHL21 in HEK293T cells apply the genomic editing to solve this problem.Owing to the features of easy-to-use,cost-effectiveness and multiple targeting ability,the CRISPR/Cas(Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated)technology become the third-generation prevalent genomic engineering tool following ZFN(zinc finger endonuclease)and TALEN(transcription activator-like effector nuclease).Bacteria and archaea have evolved sophisticated RNA-mediated adaptive immunne defense system which known as the CRISPR/Cas systems,can protcet organisms from invading viruses and plasmids.Immunity is built through acquire a short fragment of invasive DNA and integrate it into CRISPR loci,and then transcribe and processe the CRISPR loci into small interfering crRNAs(CRISPR RNAs),finally the crRNAs guide the Cas protein to complementary invading nucleic acid,which results in target interference.There are three types of CRISPR/Cas systems have been established,of which only type II CRISPR/Cas is widely used for its scalable,affordable,and easy to engineer.Type ? CRISPR/Cas consists of three genes,including Cas9 nuclease,tracrRNA(trans-activating CRISPR RNA)and pre-crRNA,which contains a nuclease guide sequences(Spacers)interspaced by Repeats.All of them with RNase? is necessary and suffcient for DNA cleavage.Spacers are transcribed and processed into crRNA,then complementary to target DNA sequence and guide Cas9 nucleases cleavage target gene.TracrRNA is bound by Cas9 and has regions of complementary to the repeat sequences in the pre-crRNA,the repeat/tracrRNA dsRNA is cleaved by RNase? to generate crRNA guides for the Cas9 nuclease.Actually,a chimeric RNA consists of crRNA and tracrRNA can direct Cas9 and form a ribonucleoprotein complex to mediates specific DNA cleavage.The PAM(protospacer adjacent motifs)is located immediately downstream of the target sequence,PAM recognition is required for CRISPR/Cas immunity.Cas9 have two nuclease domains:HNH and Ruvc,each of them cleaves one strand of the target DNA.In the presence of exogenous homologous recombination template of target DNA sequences,we can modify the target gene DNA sequence.Objective:Knock-in protein tags into the C-terminus of KLHL21 in HEK293T cells.Methods:1:Construct KLHL21 gene containing mCherry-Flag tag expressin plasmid and sgRNA sequence which targeting KLHL21 gene C terminal.2:The Cas9,sgRNA and cloned KLHL21 gene expression plasmid were transfected into HEK293T cells.3:Screening monoclonal cell lines by Blasticidin selection.4:Identification of positive HEK293T cells by PCR,Western Blot,and Immuno-precipitation.5:Construct Cre expression vector and directional deletion Blasticidin by Cre/Loxp site-specific recombination system.Results:1:Successfully construct the recombinant plasmids pKCFD.2:Successfully establishment the sgRNA vectors pHG4-21A and pHG4-21B.3:Inserted mCherry-Flag protein tags into the C-terminus of KLHL21.4:Obtain monoclonal cell lines by Blasticidin selection.5:Deleted the Blasticidin sequence by Cre/Loxp recombination system.Conclusion:In this experiment we utilize the CRISPR-Cas9 system edited the C terminal of KLHL21 genen and inserted mCherry-Flag protein tags,provide a convenience for the study of the function of KLHL21 protein establish a technical platform for studying the functions of other endogenous genes.