In vivo cloning

• In vivo cloning relies on recombinant DNA technology or genetic modification.
• in vivo cloning genes are isolated from one organism and inserted into another organism using suitable vectors , this creates a new combination of genes.
• The process involves the isolation of a gene, insertion of it into an appropriate vector, transformation of host cells with the vector containing the desired gene, selection of transformed cells that contain the correct number of copies of the gene, amplification of the gene by cell division, extraction of the gene-containing plasmid, and transfer to other cells.
• in vivo cloning can also use a bacteriophage vector , this is a virus which infects bacteria . the bacteria is infected by injecting DNA , this viral DNA will integrate itself into bacterial DNA.
• Method;

  1. Desired gene is isolated using restriction enzymes
  2. promoter regions and terminator regions are added to the genes to make sure the gene can be correctly transcribed.
  3. the gene is inserted into a vector- usually a plasmid.
  4. the recombinant plasmid is now transferred to host cells.
  5. the host cells are allowed to multiply and those that have successfully taken up the gene are identify using a marker gene.
• Promoter; tells RNA polymerase where to start transcribing mRNA

  Terminator; tells RNA polymerase when to stop.
• 3- The gene is inserted into a vector usually a plasmid:

  • cut the plasmid and the gen with the same restriction enzymes to leave sticky ends.
  • sticky ends allow the target gene to lyse with the plasmid.
  • ligase enzymes can join these two DNA fragments together they catalyze condensation reactions.
• 4. the recombinant plasmid is now transferred to host cells:

  • a solution can be used to make cell walls more permeable. Heat shock can be used to make holes in the membrane.