Hybridomas, stem cells, cloning and transgenic animals

A monoclonal antibody (mAb) is made by the descendants of a single B cell (one clone), so every molecule has the same sequence and recognises the same epitope. A polyclonal antiserum comes from many B-cell clones and recognises many epitopes. A normal B cell makes one antibody but stops dividing and dies after a short time in culture. A myeloma cell (a cancerous plasma cell) divides indefinitely; the lines used for fusion are chosen to secrete no antibody of their own. A hybridoma is a fusion of the two: it secretes the B cell's antibody and grows indefinitely.

HAT medium contains Hypoxanthine, Aminopterin and Thymidine (a common 1x working concentration is 100 µM hypoxanthine, 0.4 µM aminopterin and 16 µM thymidine). Aminopterin, a folate analogue, inhibits dihydrofolate reductase (DHFR). Without regenerated tetrahydrofolate, the de novo synthesis of purine nucleotides and of thymidylate (dTMP) stops. A cell can then make DNA only through the salvage pathways: hypoxanthine is converted to IMP by HGPRT (hypoxanthine-guanine phosphoribosyltransferase), and thymidine is phosphorylated to dTMP by thymidine kinase (TK). A cell needs both enzymes to survive in HAT medium.

Mouse mAbs given to patients provoke a human anti-mouse antibody (HAMA) response, which clears them and can cause reactions. They are therefore engineered into chimeric antibodies (mouse variable regions joined to human constant regions) or humanised antibodies (only the complementarity-determining regions, the antigen-binding loops, come from the mouse). Fully human mAbs are obtained from mice whose immunoglobulin genes have been replaced by human ones, or by phage display. Uses of mAbs include diagnostics (pregnancy test strips that detect hCG, ELISA, blood typing), affinity purification, flow cytometry, imaging and therapy.

A stem cell can divide to renew itself and can also give rise to specialised cells. It may divide asymmetrically (one daughter stays a stem cell, the other differentiates) or symmetrically (two stem cells, or two differentiating cells). Potency describes how many cell types it can produce.

Reprogramming to iPSCs: introducing four transcription factors, Oct4, Sox2, Klf4 and c-Myc, into a differentiated cell resets it to a pluripotent state. This shows that differentiation is reversible and removes the need for an embryo. c-Myc can be left out at lower efficiency, and non-integrating delivery (episomal plasmids, Sendai virus, mRNA) reduces the tumour risk.

Mouse ES cells are kept undifferentiated on a feeder layer of mitotically inactivated mouse embryonic fibroblasts, or in medium containing LIF (leukaemia inhibitory factor), which signals through STAT3. Withdrawing LIF and feeders lets them differentiate, for example into embryoid bodies. Human ES cells do not depend on LIF; they are maintained with basic fibroblast growth factor (FGF2).

Injected into an immunodeficient mouse, undifferentiated pluripotent cells form a teratoma, a benign tumour containing tissues of all three germ layers. This is a standard test of pluripotency. For mouse cells the stricter test is contribution to every tissue of a chimera, including the germ line, after injection into a blastocyst.

Applications: cell replacement therapy (haematopoietic stem cell, or bone marrow, transplantation is in established clinical use; skin grafts are another example), disease modelling with patient-derived iPSCs, drug and toxicity screening, and the study of development.

Cloning here means producing an animal whose nuclear genome is a copy of that of a donor. The method is somatic cell nuclear transfer (SCNT). The first mammal cloned from an adult somatic cell was the sheep Dolly, born in 1996.

The clone has the nuclear DNA of the nucleus donor. Its mitochondrial DNA comes almost entirely from the egg donor (a little donor-cell mitochondrial DNA can persist when the whole donor cell is fused), so the clone is not completely identical to the nucleus donor. The surrogate mother contributes no DNA, only the uterine environment. Cloning efficiency is low (Dolly was the only lamb obtained from 277 fused eggs), and many clones show abnormalities, partly because the donor nucleus must be epigenetically reprogrammed.

A transgenic animal carries a foreign gene (transgene) stably integrated in its genome and passed to its offspring through the germ line. A transgene construct usually has a promoter chosen to control where and when the gene is expressed, the coding sequence (cDNA or genomic gene, often with an intron to improve expression) and a polyadenylation signal.

Animals of the first generation (founders) are often mosaic or hemizygous (one copy of the insertion). Breeding founders and testing the offspring identifies lines with germline transmission, and crossing hemizygous parents can produce homozygous lines (1/4 of the offspring). A homozygous line can show an unexpected phenotype if the insertion has disrupted an endogenous gene (insertional mutagenesis).

Uses: studying gene function and disease models (for example the oncomouse, carrying an activated oncogene); production of human proteins in milk, where the promoter of a milk protein gene such as beta-lactoglobulin, casein or whey acidic protein directs expression in the mammary gland (a recombinant human antithrombin made in goat milk has been approved as a drug); improved livestock traits; testing vaccines and drugs; and xenotransplantation research.

A knock-out animal has a chosen gene inactivated, so its function can be learned from the phenotype. A knock-in animal has a sequence inserted or replaced at a defined locus, for example a mutant version of a gene or a reporter gene, so that it is expressed under the gene's own regulation. Both rely on homologous recombination or on genome editing.

ES cells and host blastocysts are taken from strains with different coat colours (for example agouti ES cells in a black host blastocyst). A chimera shows patches of both colours. When a chimera is mated to a black mouse, agouti pups show that ES-derived cells entered the germ line. Because the targeted ES cells are heterozygous, only about half of these agouti pups carry the targeted allele, so they are genotyped.

Conditional knock-outs use the Cre-loxP system. Cre is a site-specific recombinase from bacteriophage P1, and loxP is its 34 bp recognition site. Cre deletes the DNA between two loxP sites in the same orientation (sites in opposite orientations give an inversion instead). A mouse homozygous for a floxed allele (an essential exon flanked by loxP sites) is crossed with one expressing Cre from a tissue-specific promoter, so the gene is deleted only in that tissue. Cre fused to a modified oestrogen receptor ligand-binding domain is activated by tamoxifen, which also controls the timing. This is used when a complete knock-out is lethal to the embryo. The yeast FLP-FRT system works in the same way.

CRISPR-Cas9 speeds this up. A guide RNA directs Cas9 to a matching 20-nucleotide DNA sequence that lies immediately 5' of a PAM (5'-NGG-3' for the commonly used Streptococcus pyogenes Cas9), and Cas9 makes a blunt double-strand break about 3 bp upstream of the PAM. Repair by non-homologous end joining (NHEJ) often leaves small insertions or deletions that shift the reading frame, giving a knock-out. If a repair template with homology arms is supplied, homology-directed repair (HDR) copies the new sequence in, giving a knock-in; HDR is less efficient than NHEJ and works mainly in the S and G2 phases. CRISPR components can be injected directly into zygotes, bypassing ES cells and chimeras, though founders are often mosaic and must be bred.