Quan Flashcards
• Prokaryotes (how unique from eukaryotes)
o Lack of defined nucleus
o Poorly developed organelles
o Transcription & translation may be concurrent
o Polycistronic: referring to mRNA carrying information for the synthesis of more than one protein
• Eukaryotes (how unique from prokaryotes)
o Highly defined nucleus
o Well developed organelles
o Separate mRNA processing
o Monocistronic: one gene controls one protein
In eukaryotes, one must consider….
- DNA is in chromatin
- There are three RNA polymerases versus one
- The initial transcript requires processing before it is functional mRNA
- mRNA synthesized in nucleus and translated in cytoplasm so transcription-translation coupling does not occur
Describe Chromatin structure
- Nucleosomes
* DNA wrapped around protein, termed chromatin
4 Characteristics of Heterochromatin
- The most tightly packaged form of DNA.
- Transcriptionally silent
- Different from cell to cell
- Modification of histone tails can tighten or loosen the nucleosome
Three major Modification of Histone Proteins
- Acetylation
- Phospharylation
- Methylation
Types of Polymerases (prok and euk)
• Prokaryotes
o One kind of RNA polymerase
• Eukaryotes
o Have Polymerase II
Describe how/why RNA polymerase binds to the promoter region of DNA
- Promoter: a DNA sequence that determines the site of transcription initiation for an RNA polymerase.
- It is non-coding region
Why can either DNA strand may serve as the template?
• The promoter is asymmetric. Therefore the polymerase will always bind in the proper orientation
• It can only synthesize in the 5’ to 3’ direction.
o The template will be 3’ to 5’ direction.
Phases of mRNA synthesis
- Initiation -> Elongation -> termination
* In prokaryotes, make a ring structure that would bump the RNA polymerase
Not all promoters are alike…how so/why?
• Align 13 different σ70 promoters
o Consensus sequence at -10
o Consensus sequence at -35
o 16-18 bp between them
Describe how Sigma factors act as initiation factors
• Different sigma factors recognize different consensus promoter sequences
• Different “strength” of promoter determined by Pol interaction with promoter.
o Strong = produce more RNA
o Weaker = less amount of RNA
What happens to cause Transcriptional Termination?
• Stem-loop formation
o Presents only in prokaryotes
o Rich G-C bonds
What is transcriptional translational coupling?
- Many polymerase simultaneously transcribing the gene
- Ribosomes simultaneously translating protein
- Does not occur in eukaryotes
Prokaryotes regulate genes by repression and induction…describe the classic example of the trp operation
• Situation
o If tryptophan is present its synthesis in unnecessary
o If tryptophan is absent its synthesis is necessary.
• Under negative control
o A trans-acting repressor binds to the cis-acting operator to turn off transcription
o Repressors bind to DNA sequences operators, which overlap the promoter region.
o A bound repressor interferes with binding of RNA polymerase and transcription initiation.
What is the trp operon?
• Operon: contiguous genes transcribed as a single mRNA
• Here, all code for enzymes necessary for tryptophan (trp)
• Polycistronic:
o DNA: one promoter and five genes
The lac operon is under both negative and positive transcriptional controls…describe the situation
• Situation
o Glucose + = glucose metabolism
o Glucose +, lactose + = glucose metabolism
o Glucose -, lactose + = lactose metabolism
Decribe B-galactosidase’s role with the the lac operon
• Β – galactosidase, is the enzyme that break down lactose into galactose and glucose.
• the lac operon transcription-control region
o positive control: CAP protein
o Negative control: repressor
• Positive control of the lac operon by glucose
o CAP needed for efficient binding of pol to promoter
Definition of genetics
The study of heredity; it involves the study of cell, individuals, their offspring, and the populations within which the organisms live
Monohybrid cross
constructed by mating individuals from 2 parent strains each of which exhibits one of the 2 contrasting forms of the character under study (Pi=original parents; F1=their offspring)
Dihybrid Cross
Constructed by mating individuals from 2 parent strains when genes under study are on different chromosomes. There are 2 pairs of contrasting forms of character under study; individuals resulting from self-fertilization of the F1 generation are called F2
Mendel’s 1st Postulates
- Unit factors in pairs- genetic characters are controlled by unit factors that exist in pains in indiv. organisms
Mendel’s 2nd postulate
dominance/recessiveness - when 2 unlike unit factors for a single character are present in a single individual, one unit factor is dominant to the other which is said to be recessive
Mendel’s 3rd postulate
Segregation - during the formation of gametes the paired unit factors separate or segregate randomly so that each gamete receives on or the other with equal liklihood
Mendel’s 4th Postulate
Independent Assortment - during gamete formation segregation of pairs of unit factoes assort independently of each other (Mendel say that yellow is not always assoc with round and green not always with wrinkled)
***Not true if genes are located closely on the same chromosome
Genotype
The specific allelic or genetic contribution of an organism
Phenotype
the observable properties of an organism that are genetically controlled; segments of a DNA molecule code for expression of proteins - the function of the proteins give rise to the observed phenotypes in the organism
Allele
alternative form of a gene (unlike a unit factor)
Genetic locus
specific position or location of a gene on a chromosome
Homologous Chromosomes
the first division in meiosis separates homologous chromosomes which are not identical
Sister Chromatids
the second division in meiosis separates sister chromatids which are identical
Pleiotropy
one gene can affect more than one trait
Incomplete or partial dominance
combining gene products from the two alternative alleles produces an intermediate phenotype (ie Rr=pink in F2 generation); one factor does not dominate the other
Co-dominance
if two alleles are responsible for the production of two distinct and detectable products, the distinct detectable expression of both alleles in a heterozygote is called c-dominance (ie A, B, or AB blood types)
Polymorphism
the existance of two or more discontinuous, segregating phenotypes in a population (ie blood types)
Lethal Allels
expression or the lack of expression of certain genes can affect the survival of an organism
Gene interaction
individual characteristics are often under the control of more than one gene
- eg, epistasis = the phenomenon of masking/modifying the effects of one gene pair by the expression of another
- Novel phenotypes can be created by gene interaction
Sex-linkage
the gene that deternines a specific character is located on a sex chromosome
Cross-over
during meiosis, a limited number of crossover events occurs randomly between hologous chromosomes, the closer the two loci reside on the axis of the chromosome the less likely it is that any crossover even will occur between them - the LONGER the genetic distance, the HIGHER the chance of crossing over
DNA template strand
the strand of DNA that is copied during the synthesis of mRNA
DNA coding strand
the strand of DNA with a base sequence corresponding to the mRNA sequence and complementary to the template strand
RNA nucleotide substitution
uracil instead of thiamine
RNA sequence
- single-stranded; contains ribose and uracil (rather than deoxyribose and thymine)
- RNA is transcribed from DNA by RNA polymerase. RNA is central to protein synthesis. mRNA carries information from DNA to ribosomes. These ribosomes can read messenger RNAs and translate the information they carry into proteins.
Promoter
- a DNA sequence that determines the site of transcription initiation for an RNA polymerase
- it is non-coding
- it is asymmetric (the polymerase will always bind in the proper orientation)
- it can only synthesize in the 5’ to 3’ direction
Direction of Transcription
5’–> 3’
RNA Polymerase structure
- core enzyme consisting of five subunits: 2 α subunits, 1 β subunit, 1 β’ subunit, and 1 ω subunit
- ribonucleotide triphoshate tunnel, active site, RNA exit tunnel with flap, jaws that clamp around DNA
- function: to copy one strand of DNA into RNA
Phases of RNA synthesis
initiation- RNA polymerase bind to the promoter; sigma factor (initation factor) binds to RNA pol.
- elongation- release of sigma factor; elongation of RNA chain
- termination- formation of stem loop; dissociation of RNA from DNA template; UUUU at 3’ end
Transcription initiation site
- transcription begins with the binding of RNA polymerase to the promoter in DNA
- at the start of initiation, the RNA polymerase is associated with a sigma factor
Sigma Factor in transcription
- prokaryotic transcription initiation factor that enables specific binding of RNA pol to gene promoters
- aids in finding the appropriate -35 and -10 base pairs downstream of promoter sequences
- act as initiation factors
- different sigma factors recognize different consensus promoter sequences
- different “strength” of promoter is determined by RNA polymerase interaction with the promoter
Steps in transcription
-initiation, elongation, termination
Stem-loop structure for transcription termination
-sequence of G and C base pairings within an RNA strand followed by a series of U’s that signals the end of transcription
Trp operon regulation
ex. of gene repression; a trans-acting repressor bind to the cis-acting operator to turn off transcription
- if tryptophan is present, its synthesis is unnecessary, so the gene is repressed (off)
- if trypotophan is absent, its synthesis is necessary, so the gene is expressed (on)
Positive control in lac operon
- catabolite activator protein (CAP) protein needed for efficient binding of polymerase to promoter
- low glucose = high cAMP, lactose present = strong transcription
Negative control in lac operon
- repressor
- high glucose = low cAMP, no lactose = no transcription
Transcription and translation are coupled in prokaryotes!
- many RNA polymerases simultaneously transcribe the gene
- ribosomes simultaneously translate the protein
Operon
contiguous gene sequences transcribed as a single mRNA
Lac Operon
-The second control mechanism is a response to glucose, which uses the Catabolite activator protein (CAP) to greatly increase production of β-galactosidase in the absence of glucose. Cyclic adenosine monophosphate (cAMP) is a signal molecule whose prevalence is inversely proportional to that of glucose. It binds to the CAP, which in turn allows the CAP to bind to the CAP binding site (a 16 bp DNA sequence upstream of the promoter on the left in the diagram below), which assists the RNAP in binding to the DNA. In the absence of glucose, the cAMP concentration is high and binding of CAP-cAMP to the DNA significantly increases the production of β-galactosidase, enabling the cell to hydrolyse (digest) lactose and release galactose and glucose.
Chromatin
nucleosomes and histone proteins
Components of chromatin
DNA- exons, introns, regulatory sequences, junk
-Protein- histone, non-histone
Heterochromatin
- the most tightly packaged form of DNA
- transcriptionally silent
- different from cell to cell
Chromatin acetylation
activates transcription; associated with euchromatin
-opens up the structure so transcription can occur and genes can be expressed
mRNA is transcribed by Pol II
pol II requires general transcription factors to initiate transcription
-pol II is stimulated by distant enhancer sites
TATA Binding Protein (TBP)
a transcription factor that binds specifically to a DNA sequence called the TATA box; this DNA sequence is found about 35 base pairs upstream of the transcription start site in some eukaryotic gene promoters
Enhancer
- distant enhancer sites stimulate transcription by RNA polymerase II
- can work upstream, downstream, or in reverse orientation
- operate by DNA looping
Insulator
DNA sequences that bind specialized proteins that buffer the gene from outside effects, prevent the control region from acting outside of the domain, and prevent the spread of heterochromatin
-limit the distance that enhancers operate
Zinc finger protein
- is a transcriptional binding domain
- symmetrical structure
- needs zinc so transcriptional factors work
- small protein structural motifs that can coordinate one or more zinc ions to help stabilize their folds
Leucine zipper protein
- leucine pairs w/ itself
- structural motif in proteins found as part of a DNA-binding domain in various transcription factors
- involved in regulating gene expression.
- a super-secondary structure that functions as a dimerization domain, and its presence generates adhesion forces in parallel alpha helices
5’ capping
- to prevent RNA degradation
- a specially altered nucleotide on the 5’ end of precursor messenger RNA and some other primary RNA transcripts as found in eukaryotes.
- vital to creating mature messenger RNA, which is then able to undergo translation
- ensures the messenger RNA’s stability while it undergoes translation in the process of protein synthesis
- is a highly regulated process that occurs in the cell nucleus.
Splicing
- splicing of pre-mRNA uses snRNA, which forms the “spliceosome”
- alternate splicing of mRNA is tissue specific
- complex of specialized RNA and protein subunits that removes introns from a transcribed pre-mRNA segment
Poly A tail
- a long chain of adenine nucleotides that is added to the 3’ end of an mRNA molecule during RNA processing to increase the stability of the molecule
- a poly A site is a specific DNA sequence that cuts RNA (the AAA sequence is added on later)
Function of intercellular communication
- affects metabolism
- affects gene expression
- affects cell shape or movement
Location of receptors
- cell-surface receptors
- intracellular receptors
Forms of intracellular signaling
- contact-dependent
- paracrine
- synaptic
- endocrine
Signaling and the fate of cells
signals a cell to :
- survive
- divide
- differentiate, or
- die
Signaling complex
See diagram on pg 4 of notesThe death-inducing signaling complex or DISC is a multi-protein complex formed by members of the “death receptor” family of apoptosis-inducing cellular receptors
Signal integration
in bacterial genetics, assimilation of genetic material from one bacterium (donor) into the chromosome of another (recipient).
Ligands for intra cellular receptor
steroid hormones (estrogen, progesterone, testosterone, aldosterone, glucocorticoids, DHEA)
Domains of steroid receptors
- ligand-binding domain
- transcription-activating domain
- DNA-binding domain
Function of ionotropic receptors
-ion channel –linked receptor that mediates fast excitatory or inhibitory neurotransmission
G protein
- a family of proteins involved in second messenger cascades
- when a ligand binds a receptor, the receptor stimulates a G protein, which in turn stimulates an enzyme, which produces a second messenger
- types of G proteins:
- alpha s- stimulates adenylate cyclase
- alpha i- inhibits adenylate cyclase
- alpha q- activates phospholipase C (PLC)
cAMP
- binds to regulatory site of PKA, which releases the C (active) subunit
- is a second messenger used for intracellular signal transduction
- involved in the activation of protein kinases
PKA
- family of enzymes whose activity is dependent on the level of cAMP in the cell
- functions in the regulation of glycogen, sugar, lipid metabolism, and the cell cycle
Kinase
-a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates. The process is referred to as phosphorylation
IP3
-signaling molecule that binds to the IP3 receptor on the ER & triggers the opening of calcium channels
Ca++ activation
signaling molecule that binds to the IP3 receptor on the ER & triggers the opening of calcium channels
Tyrosine kinase receptors
- high affinity cell surface receptors for many polypeptide growth factors, cytokines and hormones
- tyrosine kinase domains come together and autophosphorylate (ligand binds and dimerizes the receptor)
Auto-phosphorylation
- addition of a phosphate to a protein kinase by virtue of its own enzymatic activity.
- tyrosine kinase receptors do this
RAS
- small G proteins for cell proliferation
- are membrane bound
- have GTP-dependent switches
- activate MAP kinases
- receptors indirectly activate RAS via SH2 signaling proteins
The MAP kinase cascade
- activated by RAS proteins
- MAP kinases subsequently activate multiple other MAP kinases
NF-kB and IkB
- NK-kB is a cell surface TNF-alpha receptor
- NF-kB is inactive in the cytoplasm with I-kB
- kinase removes I-kB
- NF-kB translocates to the nucleus
Codon
- a unit of three adjacent nucleotides along a DNA or messenger RNA molecule that designates a specific amino acid to be incorporated into a polypeptide
- the order of the codons determines the sequence of the amino acids in the protein
Reading frame
- the actual codons that are translated (read) during mRNA synthesis
- in a DNA sequence, one of three different reading frames, each of which will specify a completely different polypeptide, can be transcribed
- only 1 open reading frame in the body (b/c start and stop codons must be in the same frame)
Structure of tRNA
transfers a specific active amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation
-cloverleaf structure
Pairing between codon and anticodon
“wobble” base pairing can occur, A-T and C-G in DNA and A-U and G-C
Amino Acid Activation
Adenylation occurs and then transferred to tRNA
**by ATP
Incorporation of amino acid into a protein
peptidyl tRNA attached to C terminus of the growing peptide chain, then the tRNA molecule is freed from its peptidyl linkage, then a a new peptidyl tRNA molecule is attached to the C terminus of the growing polypeptide chain
….two step - linkage of AA to tRNA the tRNA binfs to codon in RNA
Ribosome
the decoding factory, large and small subuints
The E, P, and A sites
E= exit site P= peptidyl tRNA site A= aminoacyl tRNA sites in ribosome
Steps in translating mRNA
- ) Incoming aminoacyl tRNA docks at A site in ribosome
- ) Transferred to the P site as it becomes peptidyl tRNA
- ) Moves to exit site on the ribosome
- ) exits ribosome
How to stop translation
binding of release factor to the A site results in termination
Steps of Folding of Protein
- Folding and Cofactor binding (non-covalent interactions)
- Covalent modification by glycosylation, phosphorylation, acetylation, etc.
- Binding to other protein subunits
- Mature functional protein
Steps of Co-translational folding of a protein
- Growing polypeptide chain
- Folded N-terminal domain
- Folding C-terminal domain
- Folding of protein completed after release from ribosome
Proteasome
when proteins do not fold correctly, they go into a proteosome which degrades the protein to reusable units – think “trash can”
**chaperones help fold
Translation
Protein synthesis and degredation
Codon
3 base sequence that codes for a specific amino acid
Reading frame
start (AUG) and stop (UAG) codons frame the other codons to create proper conxext for translation
Ubiquination steps
- Ubiquitin activating enzyme
- Binding to ubiquitin ligase
- Ubiquitin ligase primed with ubiquitin
- Degradation signal on target protein
- Target protein bound to ubiquitin ligase
- 1st ubiquitin chain added to target protein
- Target protein with multiubiquitin chain…this targets protein for degredation
HIstone acytylation
most important control of histone molecules and tightness of chromatin
Pol II
-Requires general transcription factors to initiate transcription binds to the TATA sequence in promotor region with a general TF then several other TFs follow; transcription by Pol II is often stimulated by distant enhancer sites; in eukaryotes needs to be phosphorylated to start process, and then initiation factors released
General TF
Pol II uses this needed to bind Pol II to the promoter, there are 5 general factors that work for every gene
DNA-binding domain
can be classified into numerous structural types including zinc finger proteins and leucine-zipper proteins
mRNA processing steps
- 5’ capping
- Poly-A tain
- Splicing of introns
Polyadenylation
addition of many A groups to the 3’ end of the mRNA
Alternative splicing
Is tissue specific, can result in many different proteins from the same mRNA sequence
metabrotropic receptor
- extracellular
- include muscarinic achetylcholine receptors, beta-adrenogenic receptors, and others
- G-protein linked receptors and 2nd messengers
Function of G-protein
when stimulated, the G proteins dissociate and activate downstream response
Gs vs. Gi vs Gq
Gs= stimulate adenylate cyclase Gi= Inhibit adenylate cyclase Gq= activate PLC (phospholipase C)
Describe PKA and its function
PKA= protein kinase A
Function = adds phosphate to a protein;
dissociates when activated by cAMP into a regulatory and catalytic subunit
What is a kinase do?
Adds phosphate/phosphotase - removes phosphate
What is cAMP and what does it do
A secondary messenger that activates PKA….
causing it to dissociate into 2 subunits =
regulatory and catalytic
What is DAG vs. IP3? and what do each do?
-Diacyl Glyceride and IP3 (inositol triphosphate) are both secondary messengers
-DAG activates protein kinase C
IP3 is an ionotropic receptor produced by PIP (ionotronic b/c it allows Ca+ ions to flow)
Tyrosine Kinase Receptor
- extracellular
- requires a ligand to activate catalytic domain
- intracellular signaling proteins bound to phosphorylated tyrosine
Receptor dimerization
when tyrosine kinase domains come together and autophosphorylate
Ras
- small G proteins
- activated indirectly by tyrosine kinase receptors via SH2 signaling proteins
- involved in cell proliferateion
- membrane bound
- GTP dependent switches
- activate MAP kinases
MAP kinase cascade
- activated by Ras
- The upstream MAP kinases phosphorylate downstream MAP kinases
NF-kB activation
- Cytokines use this pathway
- Cell surface TNF-alpha receptor
- NF-kB is inactive in cytoplasm w I-kB
- Kinase removes I-kB (removal of inhibitory factor is what activates NF-kB)
- NF-kB translocates to the nucleus
Restiriction Enzymes
enzymes that cut the DNA sequence in specific locations
Blunt end
can allow joining of different DNA blunt cut ends
Adhesive ends
jagged ends left after restriction enzymes cut them
Matching ends
when the adhesive ends find their complement
Electrophoresis
the use of a gel and electric current to separate proteins or DNA sequences by their relative sizes
Labeling DNA
adding labeled nucleotides allows you to locate specific locations on DNA
In situ hybridization
uses labeled complementary DNA or RNA probe to localize a specific DNA or RNA sequence in a portion of a tissue
Inserting DNA into a plasmid
A circular double stranded plasmid DNA (cloning vector) undergoes cleavage with a restriction nuclease, addition of the DNA fragment to be cloned which undergoes covalent linkage by DNA ligase resulting in recombinant DNA
YAC clone
- Yeast Artificial Chromosome clone
- uses DNA ligation and yeast cell transformation to produce an artificial yeast chromosome with inserted human DNA
- Use ECORs to do very light digestion
Genomic DNA ligase
- human double stranded DNA
- ->cleaves with restriction nuclease,
- ->millions of genomic DNA fragments,
- ->DNA fragments inserted into plasmids,
- ->recombinant DNA molecules makeup genomic library
Production of cDNA
- ) Lyse cells and purify mRNA
- ) Hybridize with Poly (T) primer
- ) Make DNA copy with reverse transcriptase
- ) Degrade RNA with RNase H
- ) Synthesize a complementary DNA strand using DNA polymerase
- ) RNA fragments act as primer
- ) Double stranded cDNA copy of original mRNA
DNA sequencing
You know which nucleotide you tagged, so after running the gel you can just read the sequence from bottom up
PCR cloning
- Isolate mRNA
- Add first primer, reverse transcriptase and deoxuribonucleoside triphosphates
- Separate strands add second primer
- PCR amplification
- –> USE PCR to obtain genomic DNA or cDNA clones
Genomic DNA clones vs cDNA clones
Genomic DNA clones = introns and exons
cDNA clones = only exons
DNA fingerprinting
repeated sequences in a VNTR (variable nucleotide repeats) locus are unique to an individual….
different amounts per individual
Expression vector
- Double stranded plasmid DNA expression vector
- Cut DNA with restriction nuclease
- Insert protein coding DNA sequence behind promotor
- Introduce recombinant DNA into cells
- Overexpressed mRNA and protein
From protein to DNA
- Determine partial amino acid sequence
- Synthesize DNA probe
- screen cDNA or genomic DNA library
- gene or cDNA
- Insert into expression vector
- Introduce into E. coli or other host cell
- Protein
Tag expressed protein
- Gene for protein of interest
- Insert DNA encoding epitome tag
- Introduce into cell
- Get epitope tagged protein which can then be located using immunolocalization using antibodies to protein tag, and rapid purification of tagged proteins and any associated proteins
Fishing for interactive proteins
- Recombinant DNA techniques are used to make fusion between protein X and glutathione S transferase (GST)
- Fusion protein bound to glutathione-coated beads
- When cell extract is added, interlocking proteins bind to protein X
- Glutathione solution elutes fusion proteins that interact with protein X
The yeast two-hybrid system
- recombinant genes encoding bait and prey introduced into yeast cell (bait = known DNA binding domain plus target protein, prey = binding partner plus transcriptional activation domain)
- transcription of reporter gene and then reporter protein
Phage display
- Gene encoding a phage coat protein
- Ligation, DNA encoding peptide of interest
- Infect
- Peptide in fusion protein displayed on surface of phage
- Fusion library of many phages, each with a different peptide displayed on its surface
- Inject phage library into mouse with brain tumor
- Phage bound selectively to blood vessels of brain tumor are isolated
- Amplify in E. coli and reinject into mouse
- Phage reisolated from tumor blood vessels
- DNA that encodes a peptide that specifically binds to tumor or blood vessels
DNA foot printing
- Region of DNA protected by DNA binding protein
(will react with certain proteins) - Random cleavage by nuclease or chemical followed by removal of the protein and separation of the DNA strands
- Family of single stranded DNA molecules labed at the 5 end
- Separation by gel electrophoresis
- Foot print where no change is observed
Insertional mutation
the addition of 1 or more nucleotide base pairs
RNA - normal mRNA and intisense RNA bind to create interferon
- E. coli expressing double stranded RNA, eaten by worm
2. Double stranded RNA inserted into guy
Site-directed mutagenesis
- Plasmid cloning vector and inserted normal gene
- Strand separation
- Synthetic oligonucleotide primer containing desired mutated sequence
- Strand completion by DNA polymerase and DNA ligase
- Introduction into cells followed by replication and segregation into daughter cells
* **can create sequence that is changed at only 1 nucleotide
Gene targeting
- Embryonic stem cells growing in tissue culture
- Introduce a DNA fragment containing altered gene into many cells
- Let each cell grow to form a colony
- Test for the rare colony in which the DNA fragment fas replaced one copy of the normal gene
- Inject Embryonic Stem cells into early embryo
* **ES cells w. one copy of target gene
