IMPACT OF GENE CLONING IN HUMAN WELFARE

MICRO. – 503 (MICROBIAL GENETICS)

SUBMITTED TO:- SUBMITTED BY:-DR. Y. K. JHALA JAYVIRSINH P. SOLANKIASSISTANT PROFESSOR M.SC. AGRI. (04-2917-2016)

GENE CLONING IS THE PROCESS IN WHICH A GENE OF INTEREST IS LOCATED AND COPIED (CLONED) OUT OF DNA EXTRACTED FROM AN ORGANISM. WHEN DNA IS EXTRACTED FROM AN ORGANISM, ALL OF ITS GENES ARE EXTRACTED AT ONE TIME. THIS DNA, WHICH CONTAINS THOUSANDS OF DIFFERENT GENES.

OVERVIEW• CLONING IS A COMMON PRACTICE IN MOLECULAR BIOLOGY LABS

THAT IS USED BY RESEARCHERS TO CREATE COPIES OF A PARTICULAR GENE FOR DOWNSTREAM APPLICATIONS, SUCH AS SEQUENCING, MUTAGENESIS, GENOTYPING OR HETEROLOGOUS EXPRESSION OF A PROTEIN. THE TRADITIONAL TECHNIQUE FOR GENE CLONING INVOLVES THE TRANSFER OF A DNA FRAGMENT OF INTEREST FROM ONE ORGANISM TO A SELF-REPLICATING GENETIC ELEMENT, SUCH AS A BACTERIAL PLASMID. THIS TECHNIQUE IS COMMONLY USED TODAY FOR ISOLATING LONG OR UNSTUDIED GENES AND PROTEIN EXPRESSION. A MORE RECENT TECHNIQUE IS THE USE OF POLYMERASE CHAIN REACTION (PCR) FOR AMPLIFYING A GENE OF INTEREST. THE ADVANTAGE OF USING PCR OVER TRADITIONAL GENE CLONING, AS DESCRIBED ABOVE, IS THE DECREASED TIME NEEDED FOR GENERATING A PURE SAMPLE OF THE GENE OF INTEREST. HOWEVER, GENE ISOLATION BY PCR CAN ONLY AMPLIFY GENES WITH PREDETERMINED SEQUENCES. FOR THIS REASON, MANY UNSTUDIED GENES REQUIRE INITIAL GENE CLONING AND SEQUENCING BEFORE PCR CAN BE PERFORMED FOR FURTHER ANALYSIS.

CLONING:

• CLONING INVOLVES THE REMOVAL OF THE NUCLEUS FROM ONE CELL AND ITS PLACEMENT IN AN UNFERTILIZED EGG CELL WHOSE NUCLEUS HAS EITHER BEEN DEACTIVATED OR REMOVED.

THERE ARE TWO TYPES OF CLONING: 1. REPRODUCTIVE CLONING AFTER A FEW DIVISIONS, THE EGG CELL IS PLACED INTO A UTERUS WHERE IT IS ALLOWED TO DEVELOP INTO A FETUS THAT IS GENETICALLY IDENTICAL TO THE DONOR OF THE ORIGINAL NUCLEUS. 2. THERAPEUTIC CLONING : THE EGG IS PLACED INTO A PETRI DISH WHERE IT DEVELOPS INTO EMBRYONIC STEM CELLS, WHICH HAVE SHOWN POTENTIALS FOR TREATING SEVERAL AILMENTS.• IN FEBRUARY 1997, CLONING BECAME THE FOCUS OF MEDIA ATTENTION WHEN IAN

WILMUT AND HIS COLLEAGUES AT THE ROSLIN INSTITUTE ANNOUNCED THE SUCCESSFUL CLONING OF A SHEEP, NAMED DOLLY, FROM THE MAMMARY GLANDS OF AN ADULT FEMALE. THE CLONING OF DOLLY MADE IT APPARENT TO MANY THAT THE TECHNIQUES USED TO PRODUCE HER COULD SOMEDAY BE USED TO CLONE HUMAN BEINGS. THIS STIRRED A LOT OF CONTROVERSY BECAUSE OF ITS ETHICAL IMPLICATIONS.

INSULIN• CLONING OF HUMAN INSULIN IN A BACTERIAL HOST

    GENE SEGMENTS CORRESPONDING TO THE MATURE A & B CHAINS OF INSULIN ARE ENGINEERED INTO SEPARATE BACTERIAL PLASMIDS WITH AN ANTIBIOTIC RESISTANCE GENE AND A B-GAL STRUCTURAL GENE AS MARKERS.  THE PLASMIDS ARE SEPARATELY TRANSFORMED INTO E. COLI.  CULTURE OF THE E. COLI IN THE PRESENCE OF ANTIBIOTICS SELECTS THOSE CELLS THAT HAVE BEEN SUCCESSFULLY TRANSFORMED.  INDUCTION OF THE B-GAL GENE EXPRESSION (BY ADDITION OF B-GALACTOSE SUGAR) ALSO CAUSES TRANSCRIPTION OF THE INSULIN GENES, WHICH ARE THEN TRANSLATED BY THE BACTERIAL CELLS. THE BACTERIAL CULTURE IS DONE ON AN INDUSTRIAL SCALE TO PRODUCE THOUSANDS OF LITERS OF CLONED HUMAN INSULIN.

• FOLLOWING CHEMICAL CLEAVAGE AND PURIFICATION AWAY FROM THE PLASMID PROTEIN, THE A & B CHAINS SPONTANEOUSLY ASSEMBLE TO FORM BIOLOGICALLY-ACTIVE INSULIN. AS THIS PROCESS IN VITRO IS RELATIVELY INEFFICIENT, COMMERCIAL HUMAN INSULIN IS NOW MADE BY CLONING THE PROINSULIN MRNA (WITHOUT THE SIGNAL PEPTIDE OF PRE-PROINSULIN) IN A SINGLE HOST, AND SPECIFIC CLEAVAGE WITH THE PROTEOLYTIC ENZYMES.

BREST CANCER• BREAST CANCER RESISTANCE PROTEIN (BCRP) CONFERS

MULTIDRUG RESISTANCE TO CANCER CELLS AGAINST AGENTS SUCH AS SN-38 (AN ACTIVE METABOLITE OF IRINOTECAN), MITOXANTRONE, AND TOPOTECAN. AMONG 59 HUMAN TUMOR CELL LINES TESTED, 6 CELL LINES, A549, NCI-H460, KM-12, HT-29, OVCAR-5, AND RPMI8226, SHOWED HIGH BCRP EXPRESSION. BCRP CDNA WAS ISOLATED FROM 11 CANCER CELL LINES AND THREE VARIANT CDNAS [G34A SUBSTITUTING MET FOR VAL-12 (V12M), C421A SUBSTITUTING LYS FOR GLN-141 (Q141K), AND 944 –949 DELETION LACKING ALA-315 AND THR-316 ( 315-6)] WERE IDENTIFIED.

FUTURE THRUST• DEFECTIVE GENES COULD BE ELIMINATED• FASTER RECOVERY FROM TRAUMATIC INJURY• INFERTILITY COULD BE ELIMINATED• CLONED BODY PARTS CAN SERVE AS BACKUP SYSTEMS FOR

HUMANS• COMBAT GENETIC DISEASES• REPLICATE ANIMALS FOR RESEARCH PURPOSES & ALSO

ALTERATION OF PLANTS & ANIMALS• PRODUCTION OF PEOPLE WITH DESIRABLE TRAITS

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