Internship Report

In silico Design of Plasmid to Generate Stable Cell Line Expressing Monoclonal Antibodies with CRISPR Cas9 for Precision Gene Integration

---

Therapeutic mAbs are the class of innovative therapeutic molecules with the highest growth
(mAbs). They have great potential for the treatment of many diseases, including cancer and chronic
inflammatory conditions. In 1975 in order to develop monoclonal antibodies 2 scientists, Kohler and
Milstein developed a technique in the fusion of myeloma cells with immunized mouse immune cells
resulting in hybridoma cells capable of producing mAbs. However, this conventional method of
producing mAb is not only costly and time demanding, but it also results in a decrease in the amount
of viable antibodies because more than 99% of the cells do not survive the fusion process. The
antibodies produced through hybridoma are also not a 100% human antibody gene but a mix of
mouse or rodents which may possess immune reaction and reduced bioavailability. Over the course
there have been several ways to improve the production of monoclonal antibodies namely using
genetic vectors such as plasmid to integrate the mAb gene into the target expression system. This
could reduce production cost and also reduce toxicity and prolong bioavailability. Genome editing
technology has been to insert genetic material at specific spots into the human genome for
practically a while, this insertion ensured that the desired gene would be stably expressed in the
expression system. Here, I have designed plasmid vectors for monoclonal antibody gene insertion
into HEK293 cells using CRISPR technology. First, the AAVS1 locus was recognized as the precise
location where the mAb genes would be cloned. For this, a donor vector bearing certain HAs and an
all-in-one Cas9-expressing vector are required. As a result, a donor vector was created by fusion
cloning the mAb genes into the HA-containing, empty donor vector. Finally, gRNA spacer sequences
were cloned into the Cas9 expressing plasmid to direct the Cas9 system to cut in the required
particular location.

---

Availability

Detail Information

Series Title

-

Call Number

BM 22-028

Publisher

i3L, Jakarta : i3L, Jakarta., 2022

Collation

-

Language

English

ISBN/ISSN

-

Classification

NONE

Content Type

-

Media Type

-

Carrier Type

-

Edition

-

Subject(s)

CRISPR Cas9
Monoclonal Antibody
Stable cell line expression
PCR cloning
gene knock-in

Specific Detail Info

-

Statement of Responsibility

-

Other version/related

File Attachment

Comments

---

You must be logged in to post a comment