Selectable markers

selectable markers used in animal Biotechnology

1. Selectable markers for animal cells: Thymidine kinase (TK), Dihydrofolate reductase ( dhfr ), Chloramphenicol acetyl transferase(CAT). Presentation by : Madhwi Sharma M.Sc. Biotechnology (2 nd Year) Central University of Haryana

2. Marker genes: These are genes that help in monitoring and detection of transfection systems in order to know whether the transgene has been successfully transferred into recipient cells. Marker genes is are introduced into the plasmid along with the target gene for transfection. Two types: reporter or scorable genes selectable marker genes

3. Genes that show immediate expression in the cells/tissue resulting quantifiable phenotype. Used for analysis of gene expression and standardization of parameters for successful gene transfer in a particular technique. Detection by an assay. (protein quantification) 1. Reporter genes:

4. Selectable marker genes enable the transformed cells to survive on media containing selection agent which causes death of non-transformed cells. Examples - antibiotic, antimetabolite, and herbicide resistance genes etc. 2. Selectable marker genes:

5. de novo and salvage nucleotide synthesis pathways : De novo purine nucleotide synthesis initially involves the formation of inosine monophosphate (IMP) which is then converted into either adenosine monophosphate (AMP) or, via xanthine monophosphate (XMP), guanosine monophosphate (GMP). Source: Molecular cloning: A laboratory manual The de novo synthesis of IMP requires the enzyme dihydrofolate reductase (DHFR), whose activity can be blocked by aminopterin or methotrexate. In the presence of such inhibitors, cell survival depends salvage pathway. Cells lacking one of the essential salvage enzymes, such as HPRT, Thymidine kinase (TK) or APRT, therefore cannot survive in the presence of aminopterin or methotrexate unless they are transformed with a functional copy of the corresponding gene.

6. More information on Thymidine kinase : https://www.uniprot.org/uniprot/P04183 Thymidine kinase https://www.google.com/url?sa=i&url=https%3A%2F%2Fen.wikipedia.org%2Fwiki%2FHAT_medium&psig=AOvVaw0_RBS_BGJnqxB9wFzu4IjT&ust=1638904919866000&source=images&cd=vfe&ved=0CAsQjRxqFwoTCNjdoeDyz_QCFQAAAAAdAAAAABAU Selection of hybridomas : Hybridomas - tumor cells and plasma cells Use of thymidine kinase negative (TK−) tumor cell lines for the fusion. The thymidine kinase negative cells are obtained by growing the tumor cell line in the presence of thymidine analogs , that kill the thymidine kinase positive (TK+) cells. The negative cells can then be expanded and used for the fusion with TK+ plasma cells. After fusion, the cells are grown in a medium with methotrexate or aminopterin that blocks the endogenous pathway of nucleotide production by inhibiting the enzyme dihydrofolate reductase thus blocking the de novo synthesis of thymidine monophosphate. HAT medium - hypoxanthine, aminopterin and thymidine.

7. Antimetabolite Marker Gene - Dihydrofolate reductase ( dhfr gene): Enzyme - dihydrofolate reductase Gene - dhfr gene Inhibition - antimetabolite methotrexate. A mutant dhfr gene in mouse which has a low affinity to methotrexate has been identified. This dhfr gene fused with CaMV promoter results in a methotrexate resistant marker which can be used for the selection of transformed plants. More information on Dihydrofolate reductase : https://www.uniprot.org/uniprot/P00374 CHO cells DHFR lacking CHO cells are the most commonly used cell line for the production of recombinant proteins. Dihydrofolate reductase These cells are transfected with a plasmid carrying the dhfr gene and the gene for the recombinant protein in a single expression system, and then subjected to selective conditions in thymidine-lacking medium. Only the cells with the exogenous DHFR gene along with the gene of interest survive.

8. Chloramphenicol acetyl transferase (cat gene): Chloramphenicol acetyltransferase (or CAT ) is a bacterial enzyme that is responsible for chloramphenicol resistance in bacteria. Mechanism: This enzyme covalently attaches an acetyl group from acetyl-CoA to chloramphenicol, which prevents chloramphenicol from binding to ribosomes. Used reporter gene in mammalian cells. CAT detection by a sensitive radioactive assay the detection of the reporter gene cat. More information on Chloramphenicol acetyl transferase: https://www.uniprot.org/uniprot/P62577 CAT: Chloramphenicol acetyl transferase

9. Concern regarding use of marker genes The products of some marker genes may be toxic. ii. The antibiotic resistance might be transferred to pathogenic microorganisms. iii. A transgenic cell line with selectable marker genes can’t be transformed again by using the same selectable markers. Solutions: Avoiding selectable marker genes: S creening by an advanced technique like polymerase chain reaction. Drawback: time consuming and expensive. 2. Removal of selectable markers: use of site-specific recombinase systems to selectively excise the marker genes from the plant genome. 3. Cloning of selectable markers between transposable elements: A selectable marker gene can be cloned between plant transposable elements (Ds elements) and then inserted. The selectable marker is planked by the sequences that increase the intra-chromosomal recombination. This results in the excision of the marker gene.

10. https://www.biologydiscussion.com/genetics/engineering/selectable-marker-genes-and-reporter-genes/10752 Maniatis , T., Fritsch, E. F., & Sambrook, J. (1982). Molecular cloning: A laboratory manual . Cold Spring Harbor , N.Y: Cold Spring Harbor Laboratory. References: