• BIMS 808 4/16/03
• Pearson-White, sp3i@virginia.edu, 2-0756
• Homologous recombination gene replacement -
cont.
• RAG2 blastocyst complementation
• Tetraploid aggregation
• ES cells, in vitro differentiation
• Stem cells, stem cell therapy
• Functional genomics, knockout example

• Wanted to restrict expression to the forebrain
• Specifically to the CA1 but not CA3 or dentate gyrus pyramidal cells of the hippocampus
• Challenging because there are similar structures in functionally different regions of the brain, CA1 versus CA3
• Used a functional assay to demonstrate the effectiveness of the approach

• Promoter used was aCaMKII (alpha-calcium-calmodulin-dependent kinase II)
• Reporter had actin promoter-loxP-STOP codons-loxP-lacZNLS-polyA
• NLS (nuclear localization signal) was important to ensure transport to the nucleus were Cre/loxP excision takes place

• 11/14 founders transmitted transgene
• 9/11 tested
• 5/9 expressed, 4/9 did not express transgene
• 5 expressing mice had Cre excision specifically in the forebrain (Figs. 4, 5)
• 3/5 mice had restricted expression in CA1 region (T29-1, T40, T50)

Figure 3. Northern Blot Analysis of Cre mRNA Expression in the Adult Brain from Transgenic Lines

Figure 4. Cre/loxP Recombination Patterns in the Brains from Different Cre/lacZ Transgenic Lines Determined by X-Gal Blue Staining

Figure 5. Distribution and Efficiency of Cre/loxP Recombination in Various Transgenic Mice Examined at Higher Magnification

• Cre mRNA distribution from Northern blot (Fig. 3) appeared wider than the distribution of Cre/LoxP recombination
• However, immunohistology with Cre antibody shows staining of highest intensity in the regions where Cre/loxP recombination is highest (Fig. 6)

• Cre not expressed P16 (postnatal day 16) but detected p18 (T17 line) or p19 (T29 line) Fig. 7
• Expression onset in 3rd postnatal week was approximately the time when expression from this promoter was expected

RAG2 blastocyst complementation:

• RAG2 -/- are viable but lack T and B cells
• use these as blastocyst donors to rescue viability and study role in lymphocytes of your gene -/-
• inject your gene -/- ES cells into RAG2 -/- blastocysts
• immune system comes from your gene -/- cells
• Chen…Alt PNAS 90:4528

	When would you use tetraploid rescue? To test whether developmental arrest is due to placental defect.
	Electrofusion to make tetraploid embryos

• Extra-embryonic failure
• Embryonic death of -/- at day 8-9 can be due to placental failure, an extraembryonic defect
• Tetraploid rescue is a method to test whether the developmental arrest is due to the placental defect or an embryonic defect
• Tetraploid embryos can make extraembryonic tissues but not the embryo itself
• Electrofusion makes one cell from a 2-cell embryo, so the cells are all tetraploid
• ES cells injected into tetraploid recipient embryos make mice that are all ES-cell derived
• -/- ES cells can thus be used in combination with tetraploid embryos to complement, providing the extra-embryonic development function
• The defects that are observed in such chimeras are thus due to embryonic function of the -/- mutation
• If no defects are seen, the -/- mutant developmental defect consists entirely of placental failure.

• self-renewal
• differentiation down a pathway or lineage

• Stem cells are capable of forming most cell types
• They are not totipotent
  • i.e. they are not capable of making the whole embryo
• e.g. they donÕt make a placenta

• Embryonic stem cells (ES)
  • ES cells = pluripotent stem cells (PSC)
• Adult stem cells (AS)

• Banks of cell lines from embryos
  • many MHC genotype choices
• alter MHC genes to eliminate GVH disease
• Somatic cell nuclei to make PSC clones for cell replacement therapy for each person

ES cells can differentiate in culture, via embryoid bodies (EB)

• Allows the study of developmental processes in culture
  • influence of exogenous factors
• Studies on knockout ES cells, if target both alleles
• Studies on X-inactivation, which takes place under these in vitro conditions

In vitro differentiation of ES cells:

Hematopoietic Differentiation in EB:

• cloned by somatic cell nuclear transfer
• Adult nucleus into enucleated egg
• Nucleus from Finn Dorset into egg from Scottish Blackface
• made a viable lamb (1997) (and many nonviable lambs and embryos)
• showed that irreversible modifications don't always occur to the genome in somatic cells
• ÒAn udder way of making lambsÓ

Reproductive cloning…

• adult somatic cells from mammary gland, hence the name Dolly (Parton)
• subsequent DNA microsatellite analysis confirmed that lamb was derived from the cell population used as nuclear donor (Finn Dorset), not the recipient (Scottish Blackface)
• agreed with coat color of lambs

Distinguishing the two kinds of "cloning"

• Therapeutic cloning
  • Nucleus transferred into enucleated oocyte
  • Development to blastocyst
  • Derivation of ES cells for therapeutic uses
• Reproductive cloning
  • Nucleus transferred into enucleated oocyte
  • Development of blastocyst to newborn
  • Unethical, unsafe in humans

Immunosurgery to isolate ICM (inner cell mass):

• Hematopoietic stem cells
  • bone marrow
• Liver
• Skeletal muscle satellite cells

• Chromosome length shortened in adult
  • Even when somatic nuclei are transferred to embryos
  • Enzymes that prevent shortening are linked to carcinogenesis in adults
• Limited self-renewal in adult stem cells
• Pluripotentiality of AS cells not confirmed

Functional Genomics the next phase of the human genome project determining function of the genes gene targeting is a central part of this informatics is essential expression profiles, chips Functional genomics