recombinant DNA technologies

what is the cDNA library?

• synthesis in vitro of a double-stranded DNA complement to mRNA by reverse transcriptase and DNA polymerase

• the double-stranded in vitro synthesized DNA molecules are then cloned into a vector for amplification

• contains a collection of cDNA clones that were generated in vitro from the mRNA of a single tissue or cell population

• cDNA clone(s) of interest will be identified, isolated, and characterized

what can happen to poly(A) + RNA?

• can be separated from the other RNAs by fractionation on an oligo(dT) column

• most of RNA population is rRNA that lacks poly(A) and a small proportion is mRNA with poly(A) → poly(A) + RNA sticks to column → rRNA flows through column

what is the process of the cDNA library?

→ reverse transcription of mRNA to form cDNA: also have vectors
→ digestion with restriction enzymes, forming cleaved cDNA and vectors
→ ligation
→ insertion into E. coli
→ library amplification in rapidly reproducing bacteria
→ DNA isolation
→ collection of cDNA library of actively transcribed genes

how is RT-PCR used in cDNA cloning?

• to clone a cDNA of a particular protein whose sequence is known, one can use RT-PCR
• conversion of the mRNA to single-stranded DNA by reverse transcriptase
• a primer (based on the known sequence of that protein) is used to convert the single-stranded DNA to double-stranded
• standard PCR amplification, followed by cloning

what is the process of using RT-PCR to clone a single known gene of interest?

→ reverse primer with BamH1 site on 3' end of mRNA
→ reverse transcriptase
→ cDNA denatures, primer annealed forward
→ forward primer with HindIII site on 3' end
→ DNA polymerase
→ PCR with same 2 primers
→ cut with BamH1 and HindIII
→ ligate into BamH1 and HindIII sites of vector
→ amplified cDNA with HindIII, BamH1, and vector

what is quantitative PCR?

• to determine the quantity of individual mRNA transcripts by quantitative PCR (qPCR), or real-time PCR
• RNA is first isolated from the cell/tissue of interest
• cDNA is prepared from the RNA by reverse transcriptase
• the transcript of interest is then PCR amplified with the appropriate primers in the presence of the dye, SYBRGreen I, which fluoresces brightly when bound to double-stranded DNA
• after several PCR cycles, the C_T value (the # of cycles the fluorescence intensity exceeds the detection threshold), is inversely proportional to the # of copies of the original cDNA template

what is in a genomic library?

• chromosomal DNA from the tissue of interest is isolated and digested with a restriction enzyme, which produces a collection of DNA fragments
• an appropriate vector (DNA that can be used to carry and replicate pieces of DNA) is digested with the same restriction enzyme
• DNA ligase is used to ligate one random piece of genomic DNA into a vector
• each genomic library contains all DNA fragments in vectors

what is in a cDNA library?

• mRNA from the tissue of interest is isolated and converted to a single-stranded DNA by reverse transcriptase first, then with DNA polymerase to form a double-stranded DNA, which is then digested with a restriction enzyme or linker sequence that contains restriction enzymes
• an appropriate vector is digested with the same restriction enzyme that has the same recognition sequences as the linker
• mix the vectors with cDNA fragments, DNA ligase is used to ligate one random piece of cDNA into one vector
• all cDNA fragments will be cloned into vectors
• each cDNA fragment only contains exon(s)

what types of mutations are there?

wild type, insertion, deletion, double mutant, triple mutant

what is the molecular basis of sickle-cell disease?

• in the DNA, the mutant template strand has an A where the normal template has a T
• the mutant mRNA has a U instead of an A in one codon
• the mutant (sickle-cell) Hb has a valine (Val) instead of a glutamic acid (Glu)
• O binds poorly

what is recombinant protein expression in microorganisms?

• they are prokaryotes and unable to carry out certain post-translational modification(s)

• the most common bacterium used to produce genetically engineered proteins is E. coli

what is recombinant protein expression in fungi?

they are eukaryotic and are capable of some post-translational modification(s) and can fold some human proteins correctly

what is recombinant protein expression in insect systems?

• baculoviruses that infect insects are used to insert the DNA, which carries the coding sequences for the desired proteins to be made
• some post-translational modification(s) is slightly different in insects than it is in the mammals

what is recombinant protein expression in plants?

not all proteins can be expressed in plants, also the post-translational modification(s) are slightly different in plants than they are in mammals

what is recombinant protein expression in mammalian cell systems?

grow slower, complex nutritional needs but frequently this is the only choice for certain protein products

what is recombinant protein expression in whole animals?

• transgenic animals with a piece of "foreign" DNA, which encodes the protein of interest
• the transgenic animals are engineered to make the recombinant products in milk/eggs

why would we want to clone a gene?

• to produce large quantities of particular DNA sequences to study them in detail
• to make a large quantity of the gene's product for research or for profit
• if we use bacteria to produce the protein, product of a cloned gene, we need to use expression vectors that can yield the protein

what are expression vectors?

• designed to yield the protein product of a cloned gene in the greatest amount possible
• to optimize expression, these vectors include strong bacterial promoters and bacterial ribosome binding sites
• most expression vectors are inducible, to avoid overproduction of a foreign product that may poison the bacterial host

what is involved in the manipulation of gene expression in prokaryotes?

• the nature of the transcriptional promoter
• the ribosomal binding site
• the # of copies of the cloned gene
• the efficiency of protein translation
• the cellular localization of the synthesized protein
• the nature of the termination sequences
• the stability of the cloned protein

what is a generalized prokaryotic expression vector?

incorporates both transcriptional and translational signals for the expression of eukaryotic genes in E. coli

what are examples of eukaryotic proteins that can be produced in E. Coli from recombinant DNA?

• many hormones are normally expressed (produced) at very low levels
• preclude the ability to isolate and purify in large quantities by standard biochemical techniques
• therapeutic applications of these proteins also involve basic research on their structures and functions, which require efficient ways for producing them in large amounts at a reasonable cost
• recombinant DNA techniques that turn E. Coli cells into factories for synthesizing many low-abundance proteins (e.g. granulocyte colony-stimulating factor, insulin, GH, erythropoietin, etc,)

what is granulocyte colony-stimulating factor (G-CSF)?

• stimulates the production of granulocytes: the phagocytic WBCs that are critical to defense against bacterial infection
• produced in E. coli cells from plasmid vector containing the Iac promoter
• cancer patients need G-CSF to offset the reduction in granulocyte production caused by chemotherapeutic agents, and protect the patients against serious infection while they are receiving chemotherapy

what are the steps in producing large amounts of a low-abundance protein?

• obtain a cDNA clone encoding the full-length protein
• select the proper plasmid vector that will express large amounts of the encoded protein when it is introduced into E. Coli cells
• the key to selecting/designing such an expression vector includes: a promoter, a DNA sequence from which transcription of the cDNA can begin

what is the process of the formation of G-CSF?

→ the plasmid expression vector contains the lac promoter from E. coli and the LacZ gene
→ in the presence of the lactose analog, isopropyl β-D-1-thiogalactopyranoside (IPTG), RNA polymerase from E. coli will transcribe the LacZ gene to LacZ mRNA
→ mRNA translated to the encoded protein β-galactosidase
→ LacZ gene can be removed from the expression vector by restriction enzymes and replaced by a cloned cDNA encoding G-CSF
→ the resulting recombinant DNA can be introduced into E. coli cells
→ in the presence of the inducer IPTG, subsequent transcription from the LacZ promoter produces G-CSF mRNA, which is then translated into G-CSF protein

what is the purification of an eukaryotic protein produced in an E. coli expression system?

• to facilitate the separation of the eukaryotic protein produced in an E. coli expression system, a stretch of short nucleotide sequence is added to the end of the cDNA
• e.g. adding 6-8 histidine residues (CAU, CAC) to the C-terminus of the protein
• an affinity matrix column contains chelated nickel atoms
• proteins that are modified by the addition of histidine residues will bind to the matrix, whereas E. coli proteins will not bind to the matrix
• the bound eukaryotic proteins can be released from the nickel atoms by changing the pH in the column by adding imidazole and making the pH more alkaline
• recombinant proteins made this way are functional, and the addition of the short amino acids to either the C-terminus or N-terminus of the protein usually does not interfere with the protein's function

what is recombinant human insulin?

consists of 2 protein chains (A and B) produced from separate genes

what is the process of making recombinant human insulin?

→ coding sequences for A and B chains were cloned into 2 plasmids containing the lacZ gene: which encodes the enzyme β-galactosidase
→ recombinant plasmids were then introduced into bacterial cells and β-gal-insulin fusion proteins are made
→ affinity column containing the Ab to β-gal is used to purify β-gal-insulin fusion proteins
→ the fusion proteins are then chemically treated to separate the cloned insulin from β-gal proteins
→ purified forms of the A and B chains of insulin could be combined to form active insulin: given to diabetic patients

what is the process of expression of human insulin in E. coli?

→ transform into E. coli
→ cultured cells produce β-gal insulin fusion proteins
→ affinity column with Ab to β-gal used to purify β-gal-insulin fusion protein
→ treat with CNBr to cut protein chains from β-gal
→ refold and oxidize cysteines
→ purify for injection into humans

what are the advantages of recombinant protein production in E. coli?

• E. coli genetics are well understood
• almost unlimited quantities of protein generated
• fermentation technology is well understood

what are the disadvantages of recombinant protein production in E. coli?

• proteins contained in intracellular inclusion bodies must be disrupted
• proteins cannot be folded in ways needed for many proteins active in mammalian systems
• some proteins are inactive in humans

what protein modifications are there?

• disulfide bond formation
• proteolytic cleavage of a precursor form
• glycosylation, addition of specific sugar residues to ser, thr, or asp
• phosphorylation, acetylation, sulfation, acylation, myristylation, and palmitylation

what is a eukaryotic expression vector?

• many recombinant DNA procedures are more difficult with eukaryotic than prokaryotic cells
• most eukaryotic vectors are designed to be shuttle vectors

what is a shuttle vector?

• carries 2 types of origins of replication and selectable markers
• one functions in E. coli and another functions in eukaryotic cells
• have been developed to use in yeast, insect, and mammalian cells

what is the process of transient transfection?

1. gene of interest is cloned into an eukaryotic expression vector

2. introduction of the recombinant clone into mammalian cells by transient/stable transfection

3. in both methods, the cultured cells must be prepared first to facilitate the uptake of foreign DNA

• can be done by exposing the cells to a preparation of lipids that penetrate the membrane increasing the permeability to DNA

• or the cells will be subjected to a brief electric shock of several thousand volts called electroporation

4. recombinant plasmid DNA is added in sufficient concentration to ensure that the majority of the cultured cells will receive at least one copy

5. the eukaryotic expression vector has the origin of replication so the plasmid can be replicated in the mammalian cells to numerous copies from which the protein is expressed

6. the expression vector also has the promoter recognized by the mammalian RNA polymerase to make the mRNA of the cloned gene

7. in transient transfection, the expression plasmid does not integrate into the host chromosomes

what is the process of stable transfection?

1. the integration of the expression plasmid is facilitated by the host enzymes normally involved in DNA repair and recombination

2. because the frequency of the cells that take up a piece of foreign DNA is low, therefore, to increase the probability of such a cell population, a selection marker is used

3. a commonly used selection marker is the gene for neomycin phosphotransferase, which confers resistance to a toxic compound related to neomycin, G418

4. only cells that have integrated the expression vector into their chromosome will survive in growth media with G418

5. the integration of the expression plasmid is random thus different colonies form from the G418-resistant cells will have different rates of transcribing the cDNA

what are the transgenic animal applications of recombinant DNA technology?

• improved farm animals
• pharming
• disease models
• biopolymer
• xenografting

what are the nucleic acid applications of recombinant DNA technology?

• gene therapy
• targeted
• diagnostic probes
• vaccines
• antisense

what are the transgenic plant applications of recombinant DNA technology?

• stress-tolerant plants
• improved productivity
• therapeutic proteins
• small molecules
• vaccines

what are the recombinant microbe applications of recombinant DNA technology?

• industrial enzymes
• vaccines
• therapeutic proteins
• biopolymers
• bioremediation