The lentivirus belongs to the retroviridae family of viruses. The retroviral genome consists of a single-stranded positive-sense RNA that is converted into double-stranded DNA during replication. Unlike other viruses, viral DNA is transcribed into RNA, then translated into protein. Retrovirus RNA is reverse-transcribed into DNA and then integrated into the host cell genome. After integration, the host cell will transcribe the viral genes along with its genes, thereby producing stable transgene expression.

The lentivirus is the most common gene-delivery vehicle to establish stable cell lines. The lentivirus is a unique member of the retroviridae family as most retroviruses can not productively infect non-dividing cells, whereas lentivirus can infect cells regardless of their proliferation status. Since our design requires a stable cell line with inducible target protein expression, lentivirus is chosen as our gene carrier.

Lentivirus is a genetically modified virus that originated from HIV retrovirus. To transfer genes into the cell by retrovirus, we eliminated the disease-causing gene and preserve only genes critical to virus production. The function of each part is listed below.

Subculture

Lentivirus can not replicatand survive without the help of host’s cell. Therefore, before the production of lentivirus, culturing of packaging cell is necessarily for the following proccess.

Transfection

To produce lentiviral particle, we cotranfected three different plasmids, packaging plasmid, envelope plasmid and transfer plasmid, that are responsible for viral particle packaging, envelope formation and transfering our gene of interest respectively. After transfection of the plasmids, proteins essential for lentivrus formation should be expressed and assembled into lentivirus.

Collect supernatant & Purification

18 hours after transfection, remove media, and replace with fresh media. Wait for 24~48 hours, collect the supernatant of the culturing media which contains lentivirus.

Activation-induced cytidine deaminase(AID) is a crucial protein involved in antigen-induced B-cell activation, and differentiation occurs in the germinal center. The GC reaction is characterized by clonal expansion, class switch recombination (CSR) at the IgH locus, and somatic hypermutation (SHM) of VH genes. AID is a deaminase similar to apolipoprotein B RNA-editing cytidine deaminases(APOBEC) that carries out deamination on cytidine, which turns dG::dC into dG::dU mismatch. The dG::dU mismatch then undergoes mismatch repair(MMR) and base excision repair(BER), leading to diversification of the VH domain(SHM) or IgH region(CSR). To sum up, AID can trigger SHM and CSR, which result in the diversification of the VH and IgH domains, respectively.

As lentivirus infects hybridoma cells, the gene of transfer plasmid reverse transcript into DNA and is inserted into the genome of hybridoma. Since we designed an inducible system, the Tet-On system, in our plasmid, the expression of our target protein is regulated by the concentration of doxycycline. As doxycycline is added to the medium, it combines with rtTA and forms a complex that combines explicitly with the TetO region.

Since AID is a deaminase that would induce point mutation, the expression of AID should be tightly regulated. We incorporated the Tet-on system, an inducible protein expression system, into our design to achieve this purpose. When tetracycline is added, it combines with rtTA(reverse tetracycline-controlled transactivator) to form a rtTA-tetracycline complex, which can recognize and attach to the TetO sequence and express our target protein. By this approach, our construct can regulate the amount of AID expressed.

Internal ribosome entry site(IRES) is a joint gene engineering biobrick that recruits ribosomes to the sequence and translate without cap manners. We decided to place the IRES Biobrick before the sequence that codes the rtTA complex, which is needed to bind to TetO to drive the sequence (TetO is also known as Tet-On system promoter). A drawback of IRES sequence is that the expression of the downstream protein would be relatively lower than the specific promoter. However, it is advantageous to our project since several papers indicate that rtTA is toxic to many type of cells.[1] Our design can reduce rtTA expression when doxycycline is absent. However, while doxycycline is added, the Tet-on promoter construct will drive the entire sequence and result in a positive cycle that increases the rtTA expression and the sequence driven by the TetO promoter at the same time.[2] To sum up, our system can control both rtTA and target protein at a deficient background level when tetracycline is not added and increase the amount of rtTA and AID in a short time when tetracycline is added. On the contrary, the traditional Tet-on system would have a higher background level of rtTA and target protein when tetracycline is not added.

• Inhibit the function of AID to induce CSR

AID(wild type) plays a crucial role in both Somatic hypermutation(SHM) and Class switch reaction(CSR), which trigger point mutation in the variable region and constant region, respectively. With the DNA repair mechanism, double-strand breakage(DSB) repair, and nucleotide excision repair(NER), these mutation result from deamination caused by AID, leading to diversification of the antibody structure. However, in our project, we only want to utilize AID's function to induce SHM to increase the diversity of Ab's variable region. Research[1] shows that the catalytic domain of CSR is 517~594(172~198a.a). In order to inhibit AID from inducing CSR, we eliminate the C-terminus 16 a.a of AID.

• Avoid the off-target mutation on autosomal chromosome

According to a former study[3], AID overexpression would lead to off-target mutation on the genes other than antibody-producing gene segments. Since the catalytic domain of CSR and NES domain of the AID overlap at the C-terminus 16 a.a, we worried about the possible adverse effects on hybridoma cell that might result from the autosomal chromosome deffects. To examine whether the concept is feasible, we conducted a dry lab model to understand the effects of eliminating the NES domain of AID on the regulation of AID. As shown in Model(See Model/AID expression model Figure7, 8), the AID distribution between the nucleus and cytoplasm has little to do with the NES domain.

HA-tag is a fragment of peptide that originates from Human influenza hemagglutinin. In our construct design, we placed HA-tag after our target protein, AID, to conduct the following experiments, such as Western-blot, which confirm that our target protein is expressed.

RFP is a reporter gene widely used to ensure our construct is inserted into hybridoma and forms a stable expression clone. The confirmation of RFP expression will is carried out by fluorescent microscopy.

F2A is one of the members of the 2A self-cleaving peptide family, a class of 18–22 aa-long peptides, which can induce ribosomal skipping during the translation of a protein in a cell. F2A separates our target protein and RFP during transcription. If the sequence conjugates, it might form a fusion protein which can interrupt the process of protein folding and therefore affect the protein's function. By inserting F2A between RFP and AID, we ensure that fusion protein is less likely to be expressed, which leads to a higher possibility of properly folded AID.

Our project aims to offer an option for non-animal-derived antibodies by modifying the original procedure of hybridoma production. After AID expression, the hybridoma should secrete many different types of antibodies. For instance, it might secrete antibodies that target variant types of SARS-Covid2. Therefore, we designed a screening system to separate the hybridoma that secretes antibodies of interest from others. The following is a demonstration of our screening system.

1. Anti-CD138 added
2. Strepavidin added
3. Anti-mouse Ig added by microfluidic
4. Sorted as single clone & acquired absorption