• Biol/Micr 805 4/2/04
• Pearson-White, sp3i@virginia.edu, 2-0756
• Gene transfer
• Transgenic mice

Requirements for transgene expression Removal of plasmid sequences poison Promoter Intron polyA+ signal Enhancer

• in transient assays, this is reflected in higher proportion of transfected cells expressing the reporter
• no integration in transients
• in stable assays, there is a large variation in expression from clone to clone that cannot be explained by copy number
• probably due to differences in integration sites
• without the enhancer, transgene is more susceptible to silencing by chromosomal integration site
• some transgenes will have better penetrance than others
• position effect variegation found both in tissue culture, and in transgenic mice
• Characterized in Drosophila

• mice into which genes have been transferred
• first done in 1980
  

  three ways to transfer genes into mice microinjection into zygote nucleus retroviral infection of pre or postimplantation embryo incorporation of ES (embryonic stem) or EC (embryonal carcinoma) cells into embryo

  
  

• 4 day estrus cycle if maintained in 12:12 hour light:dark cycle
• mating ~midnight
• day of plug 0.5d (~noon)
• pseudopregnant recipient
• make pseudopregnant by plugging with vasectomized male
• estrus cycle is coordinated for implantation
• hormones, etc. primed for implantation of transferred zygotes

oviduct, preimplantation development ---> from Hogan, B., R. Beddington, F. Costantini and E. Lacy 1994. Manipulating the Mouse Embryo; A Laboratory Manual. 497 pages. Ovulation, fertilization Inverted microscope for microinjections DIC optics, egg injection Oviduct transfer

Microinjection

• 18-19 days to birth
• ~21 days to weaning
• clip tails
• isolate DNA
• test for presence of transgene
• transgene usually in every cell, including germline

• transcriptional regulatory regions as determinants of tissue-specific gene expression
• MyoD-lacZ distal enhancer
• distal locus control regions (ß-globin)
• requirement for introns
• role of oncogenes in tumor induction and progression
• self-tolerance in the immune system

Sno promoter-lacZ transgenic, E13.5

Gene expression in the a and b-globin gene clusters is controlled by common locus control regions

selective expression of novel or altered genes can be used to perturb complex systems information about development functions malfunctions e.g. SRY gene into transgenic mice showed that SRY was the testis-determining gene SRY transgene could convert female into phenotypically male mice
	Ski transgenic mouse (lower left) and non-transgenic littermate (upper right) (Sutrave and Hughes) this showed that Ski modulated skeletal muscle development and maturation

• transfer of Huntington gene exon 1 with an expanded CAG triplet repeat is sufficient to cause a progressive neurological phenotype
• used endogenous promoter (unknown)
• normal CAG6-37
• mutant CAG35-121
• encodes polyglutamine repeat in the huntingtin protein
• CAG/gln expansion in 5 other neurodegenerative diseases, all autosomal dominant
• Mangiarini et. Al. Cell 87:493-506 (1996)

• One of the first symptoms is a dyskinesia of the limbs when held by the tail. This progresses to an alternating clasping together and releasing of the feet until the mice clasp their feet together immediately after they are picked up, and can no longer release this posture.
• The mice develop a constant tremor that becomes progressively worse. The tremor tends to be less noticeable when they are quiet or asleep, but worsens under stress (for example, the removal of the cage lid) or if they reach for food or to climb out of the cage.

• tests cis elements in the transgene for proper in vivo spatial and temporal expression
• promoter-reporter constructs (+/- enhancer)
• tissue specificity
• temporal specificity
• signal transduction responses
• position effect variegation (PEV)
• transgenes land anywhere in the genome
• differences in expression depending on integration site
• different promoters/enhancers are more or less susceptible to PEV
• insulator elements protect from this effect
• Characterization is at early stage

• transgenic approaches to gene therapy
• Transgenes to make disease models
• Transgenes to correct defects
• Transgenes to test gene delivery
• protein production, e.g. in milk
• markers for chimeric or transplant studies
• 1000 copies human ß-globin transgene
• ROSA26ßgeo

• Immortalization of cells for removal to tissue culture
• SV40 T antigen
• systems for tissue-specific gene targeting
• e.g. tissue- specific promoter —Cre recombinase
• ablation of certain cell types
• diphtheria toxin (Dip-A)
• ricin (Ricin A)
• inducible cell ablation
• HSV-TK (Herpes Simplex Virus - thymidine kinase) plus gancyclovir

• tetracycline repressible system (Tet-off)
• tetracycline inducible system (Tet-on)
• ecdysone-inducible system, trinary
• lac repressor

Figure 1.   Schematic representation of lacI transgenes (A) and target transgenes (B). (A) The lacI transgenes. All constructs are driven by a 4.3-kb fragment of the human -actin promoter (shown in white; only the 3' region of the promoter is represented), containing an intron indicated by the splice donor (d) and acceptor (a) sites. For each construct, the coding region for the lac repressor is derived from segments of the wtlacI (W, shown in yellow) and synlacI (S, shown in blue) coding regions as indicated. The first four chimeras were made by exchanging the linker between the promoter and coding region, and/or the 5' part of the coding region (5'C1-4) between W and S. The next four chimeras were made by exchanging 3' segments of the coding region (3'C1-4). In the last two constructs, M (W coding region) and R (3'C4 coding region), the coding region is flanked by segments of the rabbit -globin locus (shown in red). (alt. a, potential alternative splice acceptor sites present in the lacI coding region; E and P, EcoRV and PvuII restriction enzyme sites; arrows indicate position of primers used for the PCR shown in Fig. 2). (B) The lac operator containing target transgenes. (1) SVOZ. The SV40 early promoter containing a single, symmetric lac operator drives expression of the -galactosidase (lacZ) reporter gene, which contains the endogenous Oz operator. (2) The regulatable Tyrosinase transgene (TyrlacO). Three lac operators have been introduced into the murine tyrosinase promoter. The primary operator was centered just downstream of the start of transcription by changing the endogenous promoter sequence; two additional operators were inserted 176 bp and 526 bp upstream. The modified promoter drives expression of the wt murine tyrosinase cDNA.
	Figure 6.   IPTG controls LacIR repression of the TyrlacO target gene in the mouse. Mice (A), dissected eyes (B), and cross-sections through eyes (C). Note that the nontransgenic albino and the TyrlacO, LacIR double transgenic lack pigmentation, whereas the Tyrosinase transgenic and the TyrlacO, LacIR double transgenic that has been treated with IPTG are pigmented. (ONL, outer nuclear layer; RPE, retinal pigment epithelium; Scale bar represents 25 µm in C).
	Figure 7.   Control of the TyrlacO target gene is reversible during embryogenesis and after birth. (A) Photomicrographs of embryonic eyes at E12.5. As shown in the far right panel, IPTG can cross the placenta and induce pigmentation in the TyrlacO, LacIR double transgenic RPE, just as in the adult shown in Figure 6. (EC, eye-cup edge; RPE, retinal pigment epithelium; L, lens).
(B) Photographs of P3 mice. The corresponding appearance of the eyes at birth is shown for each genotype. The mother of the double-transgenic animal shown in the panel on the far right was started on IPTG at E12.5. The eye pigmentation seen at birth shows that even after the transgene has been silenced by the lac repressor (as demonstrated by the albino phenotype of the TyrlacO, LacIR double-transgenic E12.5 eye in panel A), it can be derepressed by IPTG. (C) Tyrosinase can be silenced after a period of derepression. Left and right panels show the same TyrlacO, LacIR double transgenic animal. On the left as an infant (P8) and on the right as an adult. IPTG was discontinued at P9, causing reversion to an albino phenotype in the adult.

• eliminate position effect variegation (PEV)
• get reproducible high level expression, tissue specificity
• goes through ES cells
• can target the same site repeatedly

• gene targeting
• insertional mutagenesis
• gene traps for isolating developmentally important new genes
• dominant negative experiments
• collagen fibril biosynthesis affected using mutant mouse alpha1(I) collagen gene, perinatal lethality

• insertional mutagenesis by transgenes
• enhancer/gene traps, directly into mice
• random mutations by retroviral insertion
• enhancer/gene traps in ES cells
• random mutations in ES cells
• directed mutagenesis in ES cells

• for isolating developmentally important new genes
• For making banks of ES cells with each gene knocked out, Omnigene
• enhancer traps used in Drosophila, not so mutagenic in mice
• Weak promoter, reporter
• gene traps use splice acceptor-lacZ
• Introduction into ES cells allows for selection and testing prior to making mouse
• also introduce mutation in the gene trapped

In vitro efficiency of exon-insertion versus splice-acceptor traps
from Voss…Gruss, Dev. Dynamics 212:171 (1998)

Figure 2 |  A comparison of beta-gal, beta-geo and polyA gene-trap vectors. 

Each vector is shown inserting into intron 1 of gene X. a | The -gal vector contains a splice acceptor (SA) site immediately upstream of the lacZ reporter gene followed by a neomycin resistance (neo) selectable marker that is driven by an autologous promoter (PGK). All insertions, regardless of whether the insertion occurs in an intron (as shown) or in intergenic regions, lead to neomycin resistance and selection. If the insertion occurs in an intron, a fusion transcript is generated between the lacZ reporter and the upstream exon of gene X on transcriptional activation of the locus. b | The insertion of the -geo vector generates a fusion transcript and protein from the -geo reporter gene and the upstream exon of gene X, providing that gene X is transcriptionally active in undifferentiated embryonic stem (ES) cells. c | In this polyA-trap insertion, a fusion transcript and protein are generated from lacZ and the first exon of gene X; however, the polyA-trap also leads to a fusion transcript between neo and the downstream exons of gene X, providing a polyA site with which to stabilize the neo transcript. Several stop codons that follow neo prevent the 3' exons of gene X from being translated. The -gal and polyA-trap vectors will trap genes that are not transcriptionally active when trapping is carried out. The advantage of the polyA-trap vector over the -gal vector is that neomycin selection should only occur when the polyA-trap vector inserts upstream of a splice acceptor and a polyA site of an endogenous gene, thereby eliminating selection of intergenic insertions. (-gal, -galactosidase; -geo, -galactosidase–NeoR fusion; pA, polyadenylation; PGK, phosphoglycerate kinase 1; SD, splice donor.)

From Nature Reviews Genetics 2:756 (2001)

From Nature Reviews Genetics 2:756 (2001)

Multiple ways to abolish or reduce function of a gene product

• 1. Delete the entire gene
• most a-thalassemia mutations
• 2. Delete part of the gene
• 60% of Duchenne muscular dystrophy (DMD) mutations

e.g. Becker and Duchenne muscular dystrophy mutations

Continued…

• 3. Disrupt the gene structure
• By a translocation
  • X-autosome translocations in women with DMD
• By an inversion
  • Inversion in F8 gene
• 4. Insert a sequence into the gene
• Insertion of LINE-1 repeat into F8 gene in hemophilia A

Continued…

• 5. Inhibit or prevent transcription
• Fragile X full mutation
• 6. Promoter mutation reducing mRNA levels
• ß-globin-29(A->G) mutation
• 7. Decrease mRNA stability
• Hb-Constant spring
• 8. Inactivate donor splice site, causing read-through into intron
• Pax3 451+1(G->T) mutation

Continued…

• 9. Inactivate donor splice site, causing exon to be skipped
• Pax3 452-2(A->G) mutation
• 10. Activate cryptic splice site, causing gain/loss of coding sequence
• ß-globin intron 1-110(G->A) mutation causing Mediterranean ß-thalassemia
• 11. Introduce a frameshift in translation
• Pax3 874ins(G) mutation
• 12. Convert a codon into a stop codon
• Pax3 Q254X mutation

Continued…

• 13. Replace an essential amino acid
• Pax3 R271C mutation
• 14. Prevent post-translational processing
• Cleavage-resistant pro-insulin in familial hyperproinsulinemia
• 15. Prevent correct cellular localization of product
• ÆF508 mutation in cystic fibrosis
• 16. Dominant negative interference with complex formation
• Collagen

Dominant negative

• derived from inner cell mass of blastocysts
• supported on feeder cells
• can reinject into blastocysts
• will rejoin inner cell mass, contribute to mouse embryo, including germ line
• blastocyst injection
• or aggregation chimeras
• reimplantation into uterus

• also called homologous recombination gene targeting
• makes use of ES cells to select rare homologous recombinants
• isogenic DNA, length of flanking homology important
• positive/negative selection
• important to maintain totipotency of ES cells during selection, so will contribute to (pass through) the germline